Carbohydrate ligands specific for MHC molecules

ABSTRACT

The present invention provides a substantially purified carbohydrate ligand that specifically binds to a leczyme. The invention also provides methods to identify a carbohydrate ligand that specifically binds to a leczyme or a leczyme that specifically binds to a carbohydrate ligand. The invention further provides methods to identify a peptide that binds to the carbohydrate ligand binding site of a leczyme.  
     The present invention provides methods to isolate a carbohydrate ligand or a leczyme and to identify a carbohydrate ligand or a leczyme that modifies the function of a cell and to obtain such functionally modified cells. The invention further provides methods to modify a cell to express a carbohydrate ligand by introducing an expression vector encoding a leczyme into the cell. The invention also provides methods to modulate the immune response to an antigen by administering the antigen and a carbohydrate ligand. In addition, the invention further provides methods to treat a disease state involving a leczyme by administering a carbohydrate ligand that binds the leczyme or by administering a leczyme that has a similar binding specificity to the leczyme involved in the disease state. The invention further provides methods to diagnose a genetic basis for hemochromatosis by detecting a mutation in a class I MHC molecule that reduces it&#39;s ability to associate with β 2  microglobulin.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates generally to the field of immunologicaldisorders and, more specifically, to major histocompatibility complextransplantation molecules.

[0003] 2. Background Information

[0004] The major histocompatibility complex (MHC) codes for a variety ofgene products, many of which play a central role in the body's defenseagainst pathogenic organisms. Such molecules include the classicaltransplantation antigens and structurally related molecules, proteinsfor transport of foreign peptides within cells, serum complementproteins, the lymphokines tumor necrosis α and tumor necrosis β,cytochromes and heat shock proteins.

[0005] The classical transplantation antigens, encoded for by genes inthe MHC, are a highly polymorphic group of molecules that wereoriginally discovered for their role in determining rejection of foreigntransplanted cells and tissue. An extensive body of experimental workhas since supported a role for the classical transplantation antigens inself-recognition. In the current paradigm, transplantation antigensserve to present peptides derived from both self and foreign proteins,for recognition by cells of the immune system.

[0006] Two distinct groups of antigens, class I and class II antigens,are encoded by genes within the MHC. Class I antigens are expressed onvirtually all nucleated cells in the body and play a role in themediation of immune responses based on cytotoxic thymus-derived (T)lymphocyte mediated cell killing. Cytotoxic T lymphocytes play a role inkilling of virus infected cells and tumor cells. The class I MHCmolecule is composed of a 45 kiloDalton (kDa) heavy chain associatednon-covalently with a 12 kDa protein known as β₂ microglobulin (β₂M).The present paradigm characterizes class I antigens as presentingpeptide fragments derived from both self and foreign proteinssynthesized endogenously within the cell.

[0007] The class I molecules were discovered for their role intransplantation and were termed the “classical” class I molecules, todistinguish them from a later discovered group of class I moleculestermed the “nonclassical” class I molecules. Genes encoding thenonclassical class I MHC molecules consist of the majority of genes sofar identified in the MHC locus. Nonclassical class I MHC molecules areoverall structurally related to the classical class I MHCtransplantation antigens in having extensive sequence homology and aheavy chain noncovalently associated with β₂M. Nonclassical class I MHCmolecules are, in general, less polymorphic than the classical class IMHC molecules and are more circumscribed in their tissue distribution.Several types of nonclassical class I molecules are expressedprincipally in the gastrointestinal (GI) tract, raising questionsregarding their function, if any in the immune system.

[0008] MHC class II antigens are expressed principally by specializedantigen presenting cells in the body. Such cells are limited to theantibody producing B lymphocyte as well as macrophages and dendriticcells distributed in various tissues of the body. The class II moleculeon the cell-surface is composed of an α chain of 33 kDa and a β chain of28 kDa associated noncovalently. Class II molecules as presentlyunderstood function principally to present peptides derived from self orforeign proteins to a specialized class of T lymphocyte that supportsthe development of cytotoxic T lymphocytes, provides immunity to fungalinfections and assists B lymphocytes in the generation of protectiveantibody responses to encapsulated bacterial infections. MHC class IIantigens present peptide fragments derived from proteins taken up bycells from the surrounding environment, in contrast to classical class Imolecules, which present peptides derived from endogenously synthesizedproteins.

[0009] A variety of human autoimmune diseases have been shown to beassociated more frequently in the population with individuals whoinherit certain genes of the MHC. For many of these diseases, theassociation is localized to the region of the MHC encoding class IIhistocompatibility antigens. These diseases are not inherited by simplemendelian segregation of MHC genes, since only one sibling of a set ofidentical twins may have the disease. This feature suggests that othergenetic factors or environmental factors have roles in the developmentof autoimmunity, with genes in the MHC playing a significant part of theprocess.

[0010] The current paradigm for MHC gene function provides severaltheories to explain a role for MHC genes in autoimmune disease. Theyinclude the inappropriate expression of class II MHC molecules in cellseliciting the autoimmune response or aberrant recognition ofself-peptides by particular MHC gene products. Such theories, however,remain to be proven. In addition, the current paradigm fails to providea useful hypothesis to explain the basis for an MHC-associated ironstorage disease known as hemochromatosis. This disease is known fromanimal studies and from the genomic structure of several class I genesto involve an MHC encoded class I molecule since deletion of the β₂Mgene in these animals results in the disease.

[0011] Thus, there exists a need to develop new approaches to thetreatment of MHC associated diseases. The present invention is based ona new paradigm for the role of class I and class II antigens and otherbroadly related molecules in self-recognition and in regulation of theimmune system. This paradigm provides that self-recognition moleculeshave a central function to recognize and modify carbohydrate structures.Thus, the present invention provides new methods for identifyingcarbohydrate ligands for self-recognition molecules and utilizing suchligands to treat diseases involving aberrant self-recognition such asautoimmune diseases, inflammatory diseases or susceptibility toinfections and provides related advantages as well.

SUMMARY OF THE INVENTION

[0012] The present invention provides a substantially purifiedcarbohydrate ligand that specifically binds to a leczyme. In addition,the invention provides methods to identify a carbohydrate ligand thatspecifically binds to a leczyme or a leczyme that specifically binds toa carbohydrate ligand. The invention further provides methods toidentify a peptide that binds to the carbohydrate ligand binding-site ofa leczyme.

[0013] The present invention also provides methods for isolating acarbohydrate ligand that binds to a leczyme or for isolating a leczymethat binds to a carbohydrate ligand. The invention further providesmethods to identify a carbohydrate ligand or a leczyme that can modifythe function of a cell and to obtain such functionally modified cells.

[0014] The invention also provides methods for modifying a cell toproduce a carbohydrate ligand by introducing an expression vectorencoding a leczyme into the cell, wherein the expression of the leczymeproduces the carbohydrate ligand.

[0015] The invention also provides methods for modulating an immuneresponse to an antigen by administering the antigen and a carbohydrateligand.

[0016] The invention also provides methods for treating a disease stateinvolving a leczyme by administering an effective amount of acarbohydrate ligand that binds to the leczyme involved in the diseasestate or by administering an effective amount of a leczyme that has asimilar binding specificity to the leczyme involved in the diseasestate.

[0017] The invention further provides methods to diagnose a geneticbasis for hemochromatosis by detecting a mutation in a class I MHCmolecule that reduces it's ability to associate with β₂ microglobulin.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention results from a profound new paradigm forthe function of self-recognition molecules in organisms includingmammals. The new paradigm holds that many types of self-recognitionmolecules heretofore known as peptide recognition and presentationstructures have a more central function in the recognition andmodification of carbohydrate-based molecules. Although the currentparadigm does not exclude recognition of peptide that is bound to acarbohydrate, such as a peptide derived from a glycoprotein, the currentparadigm provides that it is the peptide rather than the carbohydratethat is bound by the self-recognition receptor molecule. Thus,self-recognition molecules of the new paradigm have the ability tospecifically bind a substrate carbohydrate structure and chemicallymodify it either by catalyzing further addition of carbohydrate or bycatalyzing chemical modification of the existing carbohydrate.Additionally, after enzymatic modification, the self-recognitionmolecule can specifically bind with equivalent or greater affinity tothe modified carbohydrate structure than to the substrate originallyrecognized.

[0019] A molecule whose function includes the enzymatic modification ofcarbohydrate and the recognition of the enzymatic product has beentermed a “leczyme” based on the combination of having bothlectin-binding and enzymatic activity in the same base molecule. Thepresent invention provides that leczyme function is characteristic ofmany peptide recognition molecules that are well known in the art. Suchmolecules include the class I and class II MHC encoded molecules andother members of the immunoglobulin gene superfamily (IgGSF) ofmolecules. In addition, leczyme function also can be associated withnonclassical class I molecules.

[0020] As used herein, “leczyme” defines a cellular protein, which cancatalyze the chemical modification of a substrate resulting in a productwith additional carbohydrate or chemically modified carbohydrate.Leczymes also can catalyze chemical modifications of a carbohydratemolecule such as phosphorylation, acetylation, carboxylation orsulfation. A leczyme can enzymatically modify other leczymes or canmodify non-leczyme molecules. In addition, a leczyme can be expressed inthe cytoplasm, on the cell-surface or can be secreted from a cell andrecognize its' enzymatically modified product either expressed on acell-surface or secreted from a cell.

[0021] A leczyme can exhibit enzymatic activity and carbohydrate bindingactivity in the same isoform of the molecule or these activities canreside separately in different isoforms of the molecule. For example,differential RNA splicing of a leczyme can result in an enzymaticallyactive isoform of the leczyme which contains a signal(s) directing theleczyme to sites an the cell normally associated with glycosylation,such as the endoplasmic reticulum or the golgi complex. Differential RNAsplicing can also result in an isoform of the leczyme that exhibitscarbohydrate recognition capability and contains a signal(s) directingthe receptor to the cell-surface or to export from the cell.Alternatively, a leczyme expressed either in the cell or on thecell-surface can contain both enzymatic activity as well as carbohydraterecognition capability in the same molecule.

[0022] Leczyme function can be resident in the groove formed at the topof MHC encoded classical class I or class II molecule, which ischaracterized in the current paradigm as a peptide-binding groove. Thenew paradigm provides that the groove functions principally to recognizea carbohydrate structure. In addition, a leczyme such as a classicalclass I or class II molecule is also endowed with the ability tocatalyze the chemical modification of the carbohydrate structure itrecognizes and to recognize the modified product.

[0023] The present invention provides compositions of substantiallypurified carbohydrate ligands that can bind to a leczyme. As usedherein, the term “carbohydrate ligand” or “ligand” means a sugar-basedmolecule where the sugar is a part of the ligand that is recognized bythe leczyme. A carbohydrate ligand can comprise one or more sugarresidues. Multiple sugar residues of a carbohydrate ligand can be linkedin either a straight chain or branched chain configuration.

[0024] Carbohydrate ligands composed of multiple sugar residues can varyin the type and location of the linkage between each residue. Sugarresidues useful for producing a carbohydrate ligand include, forexample, glucose, galactose, fucose, mannose and sialic acid. Sugarresidues of a ligand also can be acetylated, phosphorylated or sulfatedby chemically processes well known in the art. A carbohydrate ligandalso can be chemically bonded to other molecules such as a lipid,glycolipid, protein, glycoprotein, proteoglycan, glucosaminoglycan or anorganic molecule. Such additional molecules can provide the carbohydrateligand with features such as increased binding to the leczyme orincreased stability in vivo.

[0025] A carbohydrate ligand can be multivalent in nature by having morethan one carbohydrate ligand attached to a backbone structure. Thebackbone structure can be a natural protein such as a serum albumin orcan be a synthetic molecule such as a synthetic peptide. Approaches tolink multiple carbohydrate ligands to a backbone structure are known inthe art and include, for example, biotin-avidin linkage (Rothenberg etal., Proc. Natl. Acad. Sci. (USA) 90:11939-11943 (1993), which isincorporated herein by reference).

[0026] The knowledge that a self-recognition molecule is a leczyme andthat it has been selected through evolution to recognize and modify acarbohydrate structure such as a carbohydrate ligand provides newmethods to treat disease states resulting from such self-recognitionleczymes. Such disease states include, for example, autoimmunity,hemochromatosis, inflammation, transplantation rejection, andinfections. In many of the above disease states, disease results fromaberrant recognition of self-carbohydrate structures by lymphocytes.Thus, the administration of a carbohydrate ligand that can bind to theaberrant self-recognition molecule of an individual provides a means todisrupt the aberrant self-recognition cycle mediating the disease.

[0027] A variety of leczymes exist that differ in their ability tomodify particular types of molecules. This difference results fromdifferences in the specificity of the lectin binding site that leczymeshave for their substrate. Thus, a part of the leczyme structure is arecognition site for the substrate. The catalytic site of a leczyme canbe the same site as the substrate recognition site or can be a sitedifferent from the substrate recognition site. After modification of thesubstrate, the leczyme can exhibit similar or greater binding affinityfor the modified substrate over the original substrate due to coordinatebinding by both the substrate recognition site and the catalytic site ofthe leczyme or by multivalency of the ligand.

[0028] Leczymes utilized in the present invention include a broad groupof structurally related molecules, many of which are contained withinthe IGSF. The IgGSF series of genes share an evolutionary homology (ie.common ancestor) but are not necessarily functionally related,genetically linked or coordinately regulated. The products of the IgGSFhave been defined by the presence of one or more regions homologous tothe basic structural unit of immunoglobulin (Ig), known as the Ighomology unit. These units are characterized by a primary amino acidsequence of about 70-100 residues in length and include an essentiallyinvariant disulfide bridge spanning 50-70 residues in length and severalother relatively conserved residues that maintain a tertiary structureknown as the Ig fold (for review see Hunkapiller and Hood, Adv.Immunol., 44:1-63, (1989)).

[0029] The genes of the IgGSF encode many molecules with knownimmunological function, such as the immunoglobulins, T lymphocytereceptors, classical and nonclassical MHC molecules, various Tlymphocyte and B lymphocyte cell-surface molecules or β₂M. In addition,the IgGSF encodes several cell-surface molecules known to function asreceptors for cell-cell adhesion. Such adhesion molecules include, forexample, the neural cell adhesion molecule carcinoembryonic antigen.Those IgGSF molecules devoted exclusively to mediating cell adhesion orimmunological recognition such as immunoglobulins or the T cell receptorare not a leczyme.

[0030] Leczymes of he IgGSF are encoded by genes located within the MHCregion. In humans, the MHC is in a continuous stretch of DNA located onthe short arm of chromosome 6. The entire MHC in humans is called theHLA complex. In mice, the MHC is located on chromosome 17 and containsthe H-2, Q, T and M complexes. As used herein, the term “MHC-derivedgene product” means any molecule that contains at least one polypeptideencoded for by a gene located within the MHC. Leczymes that areMHC-derived gene products include class I and class II molecules. ClassI and class II molecules that are leczymes in humans are encoded bygenes within the HLA-D region such as HLA-DP, HLA-DN, HLA-DM, HLA-DO,HLA-DQ or HLA-DR, or the various alleles of HLA-A, HLA-B and HLA-C loci,or the HLA-X, HLA-E, HLA-J, HLA-H, HLA-G and HLA-F genes.

[0031] Leczymes that are class I MHC molecules contain a 45 kDapolymorphic heavy chain or α chain associated noncovalently with a smallnonpolymorphic protein called β₂M. The heavy chain is an MHC-encodedgene product located in or near the A, B or C regions of the human HLAcomplex and within or near the K or D/L regions of the Mouse H-2complex. Although β₂M is encoded by a gene located outside the MHC andon a different chromosome, the heavy chain of the class I molecule isencoded by a gene located within the MHC, thereby including a class Imolecule within the definition of an MHC-encoded gene product.

[0032] Leczymes that are class II MHC molecules are MHC-derived geneproducts composed of a 34 kDa α chain associated noncovalently with a 28kDa β chain. An additional chain called the invariant chain istransiently associated with the class II heterodimer during transport tothe plasma membrane of the cell.

[0033] Leczymes can be expressed on the cell-surface by virtue of havinga transmembrane region and cytoplasmic tail, as in the case of theclassical transplantation antigens. Leczymes also can be linked to thecell-surface in a manner similar to some nonclassical class I molecules.For example, many of the nonclassical class I Qa and Tla molecules arelinked to the cell-surface by a phosphatidylinositol (PI) linkages, andthe product of the Q10 gene appears to be secreted (Devlin et al., EMBOJ. 4:369-374 (1985)). The majority of Qa and Tla antigens lack theclassical class I cytoplasmic exons including the phosphorylation sitein exon seven (Thor et al., J. Immunol., 151:211-224 (1993)), althoughthe transmembrane domain and the seventh exon is present in Q1 and Q2gene products.

[0034] The MHC class I heavy chain is organized into three externaldomains (α1, α2 and α3), each containing about 90 amino acids each, atransmembrane domain of about 40 amino acids and a cytoplasmic anchorsegment of about 30 amino acids. β₂M is similar in size and inorganization to the external α3 domain of the heavy chain. X-raycrystallographic analysis of the extracellular portion of the MHC classI molecule shows that the α1 and α2 domains interact and are mostexternal to the cell membrane while the α3 and β₂M domains interact andare more proximal to the cell membrane. The interacting α1 and α2domains form a platform containing a deep groove or cleft located on thetop surface of the molecule.

[0035] The current paradigm for the function of the classical class IMHC molecule interprets the groove at the top of the molecule as apeptide binding site. The site is sufficiently large enough to bind apeptide of about 8-20 residues in length and present both self andforeign-derived peptides for recognition by certain T lymphoid cells.Extensive research has shown that the MHC classical class I molecule canbind peptide of about the length of the groove. In addition, the x-raycrystallographic analysis of a classical class I molecule purified froma cell indicated that a peptide was resident in the groove. However, asdescribed above, the new paradigm in the present invention provides thatthe peptide binding groove of the classical class I molecule MHC issuited for binding a carbohydrate ligand.

[0036] Leczymes that are a class II MHC molecule share significantstructural features with a class I molecule. The class II molecule is amembrane bound glycoprotein that contains external domains, atransmembrane segment, and a cytoplasmic anchor segment. The α chaincontains two external domains labelled α1 and α2 and the β chaincontains two external domains β1 and β2 domain. X-ray crystallographyshows that the α2 and β2 domains interact as a membrane proximalstructure analogous to the α3 domain and β₂M domain interaction of theclass I molecule. The α2 and β2 domains of a class II molecule thattogether form a cleft at the top of the molecule that is very similar tothe cleft formed by the α2 and α3 domains of a class I heavy chain.Extensive evidence indicates that the groove in the class II moleculecan bind and present both self and foreign peptides for recognition by Tlymphoid cells. Peptides have been isolated from the class II moleculethat are from 13-18 amino acids in length, slightly longer that theoctomeric or nonomeric peptides commonly isolated from MHC classicalclass I molecules. As discussed above, the new paradigm of the presentinvention provides that the peptide binding groove in the class II MHCmolecule, like the groove in the classical class I MHC molecule issuited for binding a carbohydrate ligand.

[0037] Leczymes also are encoded by nonclassical class I genes. In themouse, genes encoding leczymes are located in the MHC regions Q, T and Mdownstream of the classical histocompatibility antigens. There aresimilar regions in humans coding for known nonclassical class Imolecules such as HLA-F and HLA-G. The nonclassical class I genes areoverall less polymorphic than the classical class I genes and showdifferent patterns of expression. The Q, T and M complex genes of miceconsist of approximately 45 genes, coding for non-polymorphicdifferentiation antigens with limited tissue distribution.

[0038] Leczymes which are nonclassical class I MHC molecules exhibitlimited tissue distribution in comparison with leczymes that areclassical class I MHC molecules. For example, the Qa and Tla antigens,the products of the Q and T genes, are expressed on subpopulations oflymphocytes (for review, see Flaherty et al. Critical Reviews inImmunology, 10:131-175 (1990)). Previously, no convincing function hadbeen assigned to the products of the nonclassical class I genes,although they have been suggested as possible restriction elements forγδ T cells (Hershberg et al. Proc. Nat. Acad. Sci (USA), 87:9727-31(1993)). The Qa and TLa antigens have also been reported to be expressedon intestinal epithelium (Wu et al, J. Exp. Med., 174:213-218 (1991);Hershberg et al., Proc. Natl. Acad. Sci. (USA) 87:9727-97231 (1990);Wang et al., Immunogenet., 38:370-372 (1993)) where their function wasunknown. The new paradigm of the present invention provides that thesenonclassical class I molecules are leczymes.

[0039] The nonclassical class I molecule Q2, produced by a gene withinthe mouse MHC, is an example of a leczyme that is involved in irontransport (see Example I). The gene for Q2 is located in a head to headrelationship with another gene most likely encoding a mucin. Both genesshare a single promoter region, located between the genes, the promoterbeing analogous in structure to the β-globin promoter involved in ironmetabolism. The coordinated regulation of these two genes can be readilyunderstood in view of the receptor/ligand and receptor/substrateinteractions defined as leczyme function in the new paradigm.Interestingly, the Q2 gene is distinguished from other nonclassicalclass I genes in being highly polymorphic with Q2 molecules of differentstrains of mice differing significantly in amino acid sequence. Despitethese differences, the Q2 molecules from separate strains of mice allfunction as a receptor for their co-regulated gene product since, as aleczyme, Q2 can enzymatically modify it's ligand/substrate in accordancewith the lectin recognition and enzymatic function of each Q2 geneproduct and can recognize the resulting product. Thus, the combinedenzymatic/recognition capability of a leczyme as defined in the newparadigm maintains receptor/ligand relationships in the face ofextensive genetic polymorphism.

[0040] Leczymes exist with a variety of enzymatic activities. Forexample, a leczyme can have as a glycosyl transferase enzymatic activitythat results in the catalytic transfer of a glycosyl group (mono oroligosaccharide) from a glycosylnucleotide to an acceptor molecule suchas a protein, carbohydrate or lipid. However, not all glycosyltransferases are leczymes. In fact, very few such enzymes would beleczymes since the overwhelming majority of glycosyltransferases arerestricted to expression in the endoplasmic reticulum and golgi complexof the cell.

[0041] There is currently only one glycosyl transferase(β1,4-galactosyltransferase) that is previously known to be expressed inboth the cytoplasm and on the cell, for a review see. Shur, Curr. Opin.in Cell Biol., 5:854-863 (1993)). This enzyme has both carbohydraterecognition capability and carbohydrate catalytic activity and has beenimplicated in a variety of cell-cell and cell-matrix interactions. Onehallmark of the cell-surface expressed form ofβ1,4-galactosyltransferase is that it no longer retains binding activityfor the product it generates after enzymatic modification (Miller etal., Nature, 357:590-593 (1992)). Thus, this particular transferase isnot a leczyme because it fails to exhibit recognition for it's enzymaticproduct.

[0042] A Leczyme of the IgGSF can be encoded by a gene located outsidethe MHC. For example, CD-1 is a product of the IgGSF gene that isrelated in structure to the class I MHC molecule but the CD-1 heavychain is encoded by a gene outside the MHC. The T-6 CD-1 molecule isexpressed by a specialized antigen presenting cell in the skin(Langerhan's cell) and can be internalized along with MHC class IIantigen, indicating an immunological function for T-6.

[0043] The present invention provides a composition, comprising asubstantially purified carbohydrate ligand that is specifically bound bya leczyme. As used herein, the term “substantially purified” means acarbohydrate ligand that is relatively free from other contaminatingmolecules such as lipids, proteins, nucleic acids, carbohydrates orother molecules normally associated with a carbohydrate ligand in a cellor tissue. A substantially purified carbohydrate ligand can be obtained,for example, using well known biochemical methods of purification of acarbohydrate source or by chemical or enzymatic synthesis.

[0044] A carbohydrate ligand of the present invention can include knownforms of carbohydrate containing molecules such as glycoproteins,proteoglycans, glycolipids or mucopolysaccharides that have N-linked orO-linked forms of glycosylation. The proteoglycans include, for example,mucins and those proteoglycans glycosylated with hyaluronate,chondroitin sulfate, heparin, heparan sulfate or dermatin sulfate.Glycolipids that contain carbohydrate ligands include, for example,acylglycerol, a sphingoid or a ceramide.

[0045] A sample containing a carbohydrate ligand can be obtained from avariety of sources such as from fluids, tissues or cells. These sourcescan be from any plant species or any animal such as a mammal or anyorganism. A source of carbohydrate ligand can also include a cell thathas been modified by introducing into the cell an expression vector thatencodes a leczyme or a protein that when expressed contains acarbohydrate ligand.

[0046] A sample containing a carbohydrate ligand can be obtained from achemically produced library of carbohydrates. Such libraries can be madeby mixing carbohydrates from natural sources and fromenzymatically-produced sources. In addition, individual carbohydratesfrom the library can be tagged with a detectable label such as afluorescent label to assist in structural determination of thecarbohydrate ligand.

[0047] A sample containing a carbohydrate ligand can be processed tofurther purify the ligand by methods well known in the art. Such methodsinclude, for example, purification of glycoconjugates, labelling Ofglycoconjugates by chemical or metabolic means, release ofoligosaccharides from glycoconjugates and characterization of thestructure of the released carbohydrate (see, for example, Ausabal et al,In Current Protocols in Molecular Biology Vol. 2, chapter 17, (GreenPublishing Associates and Wiley Interscience, New York, 1994); Fukudaand Kobata, Glycobiology: A practical Approach, (IRL Press, New York,1993), both of which are incorporated herein by reference). In addition,these methods are useful for structural characterization, includingsequencing of the carbohydrate ligand. Elucidation of the structure of acarbohydrate ligand purified from a tissue or a cell can enable futureproduction or the ligand by direct chemical synthesis or enzymaticsynthesis or purification from a natural source.

[0048] The present invention provides methods to identify a carbohydrateligand that can bind to a leczyme. In this method, a sample containing acarbohydrate ligand is contacted with a leczyme suspected of binding tothe ligand under suitable conditions to allow specific binding of theligand to the leczyme. Suitable conditions include, for example, anappropriate buffer concentration and pH and time and temperature thatpermits binding of the particular leczyme and the carbohydrate ligand.After a suitable reaction period, the amount of carbohydrate ligandbound to the leczyme can be determined, for example, by attaching adetectable moiety such as a radionuclide or a fluorescent label to thecarbohydrate ligand and measuring the amount of label that is associatedwith the leczyme after any unbound carbohydrate ligand has been removedfrom the ligand-leczyme complex.

[0049] As used herein, “detectable label” means a molecule whosepresence can be detected due to a physical, chemical or biologicalcharacteristic of the molecule. Detectable labels include, for example,radioisotopes, fluorescent molecules, enzyme/substrate systems, orvisually detectable molecules. Methods for detectably labelling acarbohydrate molecule are well known in the art, and include, forexample, reduction with NaB(³H)₄ or synthesis with radiolabelled sugars(see, for example, Varki, surpa, 1994 and Rothenberg et al., Proc. Natl.Acad. Sci. (USA), 90:11939-11943 (1993), both of which are incorporatedherein by reference, and Fukuda and Kobata, supra 1993). In addition,kits for the preparation of a labelled carbohydrate molecule are readilyavailable from commercial sources such as Oxford GlycoSystems (Rosedale,N.Y.).

[0050] Methods to remove unbound labelled ligand from the ligand-leczymecomplex depend, for example, on attaching the leczyme to a solidsupport. Solid supports useful in the present invention and methods toattach proteins to such supports are well known in the art (see forexample Harlow and Lane, Antibodies: A laboratory Manual (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), which isincorporated herein by reference). Such solid supports include, forexample, Sepharose, agarose or polystyrene.

[0051] After a suitable reaction period and after any unbound label hasbeen removed from the support by, for example, washing, the amount oflabel attached to the solid support provides a direct measurement of theamount of carbohydrate ligand bound to the leczyme on the support.Alternatively, the amount of labelled carbohydrate ligand bound to thesupport can be indirectly determined after the reaction period bymeasuring the amount of unbound label and subtracting this from theamount of label added at the start of the reaction.

[0052] To accurately determine the amount of labelled ligand that bindsspecifically to the leczyme, a control reaction can be performed whereall conditions are the same as in the binding reaction between thelabelled ligand and leczyme except that the leczyme is not included inthe control reaction or the leczyme is replaced by an irrelevant proteinsuch as a serum albumin. The control reaction determines the amount ofbinding of the labelled carbohydrate ligand that occurs nonspecificallysuch as the amount of labelled ligand that binds to the solid supportrather than to the leczyme on the solid support. Thus, it is necessaryto subtract the nonspecific binding value obtained from the controlreaction from the binding value obtained from the reaction that includedboth the labelled carbohydrate ligand and the leczyme to determine theamount of ligand that specifically bound the leczyme under theconditions tested.

[0053] An advantage of using a solid support is that the labelled ligandcan be added in excess relative to the leczyme, making it possible toidentify lower levels of binding affinity between the carbohydrateligand and the leczyme. Methods such as Scatchard analysis are wellknown in the art for determining the binding affinity between twomolecules, both of which can be in solution or one of which can beattached to a solid support. Equilibrium dialysis is an example of amethod where the binding of a ligand to leczyme can be determined whenboth molecules are in solution.

[0054] Methods to measure the binding of a labelled carbohydrate ligandto a leczyme also can be performed when the leczyme is associated with acell. In a manner analogous to the use of solid supports, cells thatexpress the leczyme on the cell-surface can bind the labelledcarbohydrate ligand and, after a suitable reaction period, the cells canbe separated from the unbound ligand by methods well known in the artsuch as by centrifugation or filtration. Cells that express a leczyme inthe cytoplasm can also be used to detect binding of a carbohydrateligand to the leczyme provided the cell membrane has been sufficientlypermeabilized to allow access of the carbohydrate ligand to the leczymein the cell. Methods that use cells in binding assays such asantigen-antibody binding assays are well known in the art (see, forexample, Harlow and Lane supra, 1988) and are generally applicable tobinding assays between a carbohydrate ligand and a leczyme.

[0055] A leczyme-expressing cell can be a cell that naturally expressesthe leczyme such as a lymphocyte that expresses a class I or class IIMHC encoded leczyme or can be a cell that expresses the leczyme as aresult of introducing an expression vector encoding the leczyme into thecell. Leczyme-expressing cells can be obtained from in vivo sources bymethods well known in the art such as mechanical disruption of tissue ordigestion of tissue by enzymes to release cells from their surroundingmatrix (see for example, Freshney Culture of Animal Cells (Alan R. Liss,New York, 1993), which is incorporated herein by reference). Aleczyme-expressing cell can be a cell line that is available from publiccell repositories such as from the American Type Culture Collection.

[0056] It is well known in the art that the binding between twomolecules can be performed when either of the two molecules contains adetectable label. Thus, the identification of a detectably labelledcarbohydrate ligand that binds to a leczyme attached to a solid supportor a cell also can be performed if the leczyme contains the detectablelabel and the carbohydrate ligand is attached to a solid support orexpressed by a cell. A leczyme can be detectably labelled using methodsfor labelling a protein, which are well know in the art and include, forexample, biotinylation or incorporation of radioisotopic labelledprecursors. A carbohydrate ligand-expressing cell can be a cell obtainedfrom tissues or organs or can be a cell line such as a cell lineavailable from a public repository.

[0057] Methods for attaching a carbohydrate ligand to a solid supportdepend on the chemical nature of the ligand. Thus, attachment can beaccomplished through the carbohydrate moiety or other molecule bonded tothe carbohydrate ligand attachment via chemistry suitable for attachingcarbohydrate, peptide or lipid structures to a solid support. Methods toattach carbohydrates, proteins or lipids to various types of solidsupports are well known in the art.

[0058] The binding of a carbohydrate ligand to a leczyme can bedetermined without the need for a detectable label by measuring aphysical characteristic of the either the ligand or the leczyme such asabsorption of ultraviolet radiation. Such methods for quantitating aprotein or carbohydrate by physical characteristics are well known inthe art. The ability to follow a physical characteristic of the ligandor leczyme can be applied to binding assays that use a solid support oran expressing cell or when both molecules are in solution. The bindingof a carbohydrate ligand to a leczyme also can be evaluated if theligand is a substrate for the enzymatic activity of the leczyme. In thiscase, binding can be measured by following substrate conversion kineticsmeasured, for example, by the Michealis-Menten equation (Devlin,Textbook of Biochemistry (Wiley-Liss Inc. New York, 1992), which isincorporated herein by reference).

[0059] Methods for identifying a carbohydrate ligand that binds aleczyme can be performed using a single purified carbohydrate ligand ora limited number of carbohydrate ligands, which can be purified byconventional procedures as described above or can be purified by bindingto a reagent. A purified carbohydrate ligand can also be detectablylabelled by methods disclosed herein. A carbohydrate ligand that is notpurified, such as one that is in a sample containing other molecules,can be used in a binding assay provided it is attached to a solidsupport or is expressed by a cell and binding is determined by detectingbinding of a leczyme. In this case, if the non-purified carbohydrateligand can bind the leczyme, the sample containing the ligand can besubjected to purification and subsequent binding assays to obtain thecarbohydrate ligand in a purified state.

[0060] Purified leczymes can be obtained from cells by classical methodsfor protein or glycoprotein purification such as methods known in theart for purifying class I or class II molecules. Leczymes also can beobtained from cells that have been modified by molecular biologicaltechniques to enable expression of a leczyme. A gene encoding a leczymecan be cloned into an expression vector and then introduced into a hostcell. Vectors are well known in the art and include, for example,cloning vectors and expression vectors, as well as plasmids or viralvectors (see, for example, Goedell, Methods in Enzymology, vol. 185(Academic Press, New York, 1990), which is incorporated herein byreference). A baculovirus vector is an example of a vector that can beused to express a leczyme in insect cells and result in expression ofnew carbohydrate ligands on the cell.

[0061] A vector comprising a nucleic acid molecule encoding a leczymealso can contain a promoter or enhancer element, which can beconstitutive or inducible and, if desired, can be tissue specific. Hostcells also are known in the art and an appropriate host cell can beselected for the particular vector to be used. For example, abaculovirus transfer vector can be used with baculovirus DNA to infectinsect cell lines such as SF21 cells. Cloning of such transformed cellsto produce a stable cell line can provide a source of the expressedleczyme or can provide a source of carbohydrate ligand modified by theexpressed leczyme.

[0062] The gene encoding a leczyme can be expressed as a fusion proteinto assist in purification or in further downstream Processing of theleczyme. For example, the leczyme can be produced as a chimeric proteinfused to the CH2 or CH3 domain that constitutes the Fc binding region ofan immunoglobulin molecule, as was performed previously for expressingthe CD22β lectin (Stamenkovic et al. Cell, 66:1133-1144 (1991)). The useof Protein A from Staphylococcus aureus bound to a solid support, whichis readily available from commercial sources, can be used to purify theFc containing chimeric leczyme. In addition, the solid supportcontaining the chimeric leczyme can be used directly to evaluate bindingof a carbohydrate ligand.

[0063] The present invention provides methods to identify a leczyme thatbinds a carbohydrate ligand. In this method, a sample containing aleczyme is contacted with a carbohydrate ligand suspected of binding tothe leczyme under suitable conditions to allow specific binding of theligand to the leczyme. The methods that have been described above foridentifying a carbohydrate ligand that binds to a leczyme can also beused to identify a leczyme that binds to a carbohydrate ligand. Leczymesto be identified for binding include, for example, a purified leczyme ora leczyme contained within a complex mixture such as a mixture ofproteins expressed from a cDNA expression library. Methods to produce acDNA expression library are well known in the art (see, for example,Sambrook et al, Molecular Cloning: A laboratory manual (Cold SpringHarbor Laboratory Press 1989), which is incorporated herein byreference).

[0064] The present invention provides methods of purifying acarbohydrate ligand that specifically binds to a reagent. In thesemethods, a sample containing the carbohydrate ligand is contacted withthe reagent under suitable conditions to allow formation of aligand-reagent complex. Suitable conditions includes, for example, anappropriate buffer concentration and pH and time and temperature thatpermits binding of the carbohydrate ligand to the reagent. Theligand-reagent complex is then separated from the rest of the sample bya separation method such as by washing, and the ligand is dissociatedfrom the complex.

[0065] As used herein, “reagent” means a chemical or biological moleculethat can specifically bind to a carbohydrate ligand. For example, aleczyme that binds to a carbohydrate ligand is a reagent that can beused to purify that ligand. Also, an antibody can be a reagent if it canreact specifically with the carbohydrate, protein or lipid portion of acarbohydrate ligand.

[0066] Purification of the carbohydrate ligand can be accomplished ifthe reagent is attached to a solid support such as agarose, Sepharose orplastic. Methods for coupling a protein or a carbohydrate to a solidsupport, disclosed above for detecting the binding of a carbohydrateligand to a leczyme, also are useful for attaching a reagent to a solidsupport.

[0067] Methods to dissociate a carbohydrate ligand from a ligand-reagentcomplex can depend on the nature of the reagent. For example, if thereagent is a leczyme, then a method for dissociating the complex caninvolve competitive inhibition of the complex with a sugar structurethat has binding affinity for the same site in the leczyme that bindsthe carbohydrate ligand. Other well known treatments that are useful fordissociating a carbohydrate ligand from a reagent include, for example,extremes in pH, high salt concentration or chaotrophic agents (see, forexample, Harlow and Lane, supra, 1988), which is incorporated herein byreference and Varki, supra, 1994). Carbohydrate ligands purified by theabove disclosed methods are suitable for structural analysis asdescribed above, in order to enable future production of the ligand bychemical or enzymatic synthesis.

[0068] An antibody that specifically binds to a carbohydrate ligand canbe produced to the carbohydrate or a protein moiety or a lipid moiety,if such moieties are bonded to the ligand. An antibody specific for thepeptide backbone of carbohydrate ligand such as the peptide backbone ofa mucin can be useful for purifying a source of mucin from differentcells or from different individuals, since the peptide backbone can bemore conserved between peptide containing carbohydrates than thecarbohydrate portions of these molecules. Methods for producingantibodies such as polyclonal antibodies, monoclonal antibodies,antibody fragments or the like, that are specific for protein,carbohydrate or lipid are well known in the art (see, for example,Harlow and Lane supra, 1988).

[0069] The present invention provides methods for purifying a leczymethat specifically binds to a carbohydrate ligand. In these methods, asample containing the leczyme is contacted with a carbohydrate ligandunder suitable conditions to allow formation of a ligand-leczymecomplex. Suitable conditions includes, for example, an appropriatebuffer concentration and pH and time and temperature that permitsbinding of the leczyme the carbohydrate. The ligand-reagent complex isthen separated from the rest of the sample by a method such as bywashing, and the leczyme is dissociated from the complex.

[0070] Purification of the leczyme can be accomplished if thecarbohydrate ligand is attached to a solid support such as agarose,Sepharose or plastic. Methods for coupling a carbohydrate ligand to asolid support, such as those disclosed above for detecting the bindingof a carbohydrate ligand to a leczyme, are useful for attaching acarbohydrate ligand to a solid support. Methods for dissociating theleczyme from the ligand-leczyme complex can utilize the methodsdisclosed herein for dissociating a carbohydrate ligand from aligand-leczyme complex.

[0071] The present invention provides methods to identify a carbohydrateligand that modifies the function of a leczyme-expressing cell bycontacting a sample containing a carbohydrate ligand with the cell undersuitable conditions, which allow specific binding of the ligand to theleczyme on the cell. After a suitable period of time to allow forbinding of the ligand to the leczyme, the cells are evaluated todetermine their function. A carbohydrate ligand that modifies thefunction of a leczyme-expressing cell is one that when contacted withthe cell results in a function that differs from the function of thesame type of cell that had not contacted the ligand.

[0072] As used herein, “function” in reference to a cell includes anyactivity that can be detected for a cell. The function of a cell canvary with the nature of the cell in question. For example, the functionof a T lymphocyte can include activities such as the production ofcertain cytokines, acquisition of cell mediated lympholysis, ability tomediate antibody dependent cell mediated cytotoxicity or the ability tohelp B lymphocytes to produce antibody. Thus, a particular carbohydrateligand that can bind to a leczyme on a T lymphocyte and subsequentlyeffect the function of the cell can do so by increasing or decreasingany of the above T lymphocyte functions.

[0073] Contacting a carbohydrate ligand with a leczyme-expressing cellcan be performed in vitro in a cell culture medium. Methods formeasuring the function of lymphoid cells or other cells are well knownin the art (see for example, Colligan et al., Curr. Protocols inImmunol. (Greene Publishing Associates and Wiley Interscience, New York,1992); Mishell and Shiigi, Selected Meth. in Cell. Immunol. (W. H.Freeman and Co., New York, 1980), each of which are incorporated hereinby reference)

[0074] The present invention also provides methods to identify a leczymethat modifies the function of a carbohydrate ligand-expressing cell.Methods described above for identifying a carbohydrate ligand thatmodifies the function of a leczyme-expressing cell are also useful foridentifying a leczyme that modifies the function of a carbohydrateligand-expressing cell.

[0075] The present invention provides methods to modify is the functionof a leczyme-expressing cell by contacting the cell with a carbohydrateligand that binds the leczyme. In addition, the invention providesmethods to modify the function of a carbohydrate ligand-expressing cellby contacting the cell with a leczyme that binds the ligand. Theidentification of either a carbohydrate ligand or a leczyme that canmodify the function of a cell has both in vitro and in vivo uses. Forexample, ligands or leczymes capable of decreasing or increasing thefunctional activity of cell that is involved in a disease state can beadministered to an individual to treat the disease.

[0076] The present invention provides methods to identify a peptide thatcan bind to the carbohydrate ligand binding-site of a leczyme. Thesemethods involve contacting a sample containing a peptide or peptides tobe tested with a leczyme under suitable conditions to enable binding ofpeptide to the leczyme. Subsequently, the leczyme is reacted with acarbohydrate ligand known to bind to the leczyme. The reaction isperformed under conditions suitable for the carbohydrate ligand to bindto the leczyme. Alternatively, the peptide, leczyme and carbohydrateligand can be added together at the start of the reaction.

[0077] The carbohydrate ligand can be added directly to the mixturecontaining the peptide and leczyme or can be added after any unboundpeptide has been removed from the leczyme. After the end of thereaction, the amount of carbohydrate that bound to the leczyme isdetermined and compared to the amount of carbohydrate ligand that boundto leczyme in a control sample that did not contain peptide. If theamount of carbohydrate ligand that bound to the leczyme in the samplecontaining peptide is less than the amount of carbohydrate ligand thatbound to the leczyme in the control sample, then it can be concludedthat the peptide had bound to the carbohydrate ligand binding site ofthe leczyme and is therefore a peptide mimetope of the carbohydrateligand.

[0078] A peptide mimetope can be identified in an assay format thatutilizes a carbohydrate ligand containing a detectable label and aleczyme that is bound to a solid support or is expressed by a cell.Methods disclosed herein for identifying a carbohydrate ligand that bindto a leczyme are useful to generate the assay format for identifying apeptide mimetope of a carbohydrate ligand.

[0079] A defined peptide sequence can be chemically synthesized orproduced by biological methods, such as by recombinant DNA techniques(see, for example, Sambrook et al., supra, 1989). A complex mixture ofpeptides also can be used to identify a peptide mimetope. Such complexmixtures can include, for example, a mixture of defined sequences, orcan be a semi-random or random library of sequences. Methods to generatepeptide libraries by such methods as chemical synthesis on a bead or amicrotiter plate or biological production such as on the surface of abacteriophage are well known an the arc (see, for example, Huse et al.,Science 246:1275-1281 (1989), which is incorporated herein byreference).

[0080] A peptide that can bind to the carbohydrate ligand binding siteof a leczyme can also have some of the functional characteristics of acarbohydrate ligand and thus be considered a functional mimetope of thecarbohydrate ligand. Such peptide mimetopes can be used to modify thefunction of a cell and also can be used to treat a disease state thatinvolves a leczyme that can bind to the mimetope.

[0081] The present invention provides methods to modify a cell toproduce a carbohydrate ligand, comprising introducing an expressionvector encoding a leczyme into a cell to obtain expression of theleczyme, which results in production of the carbohydrate ligand by thecell. Cells producing a particular carbohydrate ligand are useful toprovide unique types of ligands, which can be purified from the cells.In addition, such cells are useful in binding assays to identify aleczyme that binds the ligand.

[0082] The present invention provides methods for modulating an immuneresponse in an individual, such as a human or other animal, using anantigen for which the immune response is desired and a carbohydrateligand that binds to a leczyme. As leczymes include, for example, themajor histocompatibility complex molecules, that are involved inpresentation of foreign molecules for recognition by cells of the immunesystem, injection of a carbohydrate ligand and an antigen can modulatean immune response. A used herein, “modulate” means increase ordecrease. An increase in the immune response can be obtained byadministering a carbohydrate ligand bound to antigen such chat theantigen is targeted via the leczyme to an antigen presenting cell.

[0083] An antigen can be associated with a carbohydrate ligand bycovalently bonding the antigen to carbohydrate or to any protein orlipid of the ligand using methods well known in the art. The actualmethod to covalently couple the antigen to the carbohydrate ligand willdepend on the nature of each molecule to be coupled and whether thecoupling procedure is detrimental to the any critical antigenicdeterminants of the antigen or the capability of the carbohydrate ligandto bind its target leczyme. Such detrimental effects can be readilyevaluated in binding assays as described above.

[0084] More than a single antigen molecule or more than a singlecarbohydrate ligand can be coupled together to produce an immunogen.Such molecules can be made multivalent for either or both of the antigenor the carbohydrate ligand and can be used for eliciting a greaterimmune response than an immunogen containing a single molecule ofantigen and a single molecule of a carbohydrate ligand.

[0085] Methods to increase an immune response in an individual are wellknown to those in the art and require optimization of parameters such asdose, route of administration, use of an adjuvant, or schedule ofadministration (see, for example, Harlow and Lane, chapter 5, supra,1988). An increased immune response obtained after administering anantigen and a carbohydrate ligand is achieved when the immune responseparameter has increased by a statistically significant level over thelevel of the parameter manifested prior to administration of the antigenand carbohydrate ligand.

[0086] The immune response parameters that can increase afteradministering an antigen associated with a carbohydrate ligand includean antibody-mediated response or a cellular-mediated response. Methodsto measure antibody immune responses are well known to those in the artand include, for example, detection of immunoglobulins by both in vitroand in vivo methods (see for example Harlow and Lane, supra, 1988).Methods to measure cellular immune responses are also well known in theart and include in vivo methods such as skin testing for delayedhypersensitivity and in vitro methods such as direct cell cytotoxicityor cell activation assays (see, for example, Coligand et al. supra,1992; Mishell and Shiigi, supra, 1980).

[0087] An antigen associated with a carbohydrate ligand can be used todecrease an immune response to the antigen and can be particularlyuseful for treating a deleterious immune response such as an autoimmunedisease state. Methods for decreasing an immune response can, under someconditions result in a prolonged state of specific immunologicalunresponsiveness to the antigen, commonly referred to as a state oftolerance to the antigen.

[0088] Decreasing an immune response to an antigen by administering theantigen bonded to a carbohydrate ligand can be accomplished usingmethods well known in the art to suppress or tolerize an individual toan antigen. Such methods include, for example, administration of lowdoses, monomeric and nonaggregated forms of the antigen and carbohydrateligand or administration orally. In addition, a decreased immuneresponse can be obtained by administering the antigen and carbohydrateligand concurrently with an immunosuppressive agent such as cyclosporinA, FK506 or antibodies to a particular T lymphocyte cell-surfacereceptor. Methods for using such agents to decrease the immune responseto an antigen in humans or animals are well known in the art.

[0089] The present invention provides methods for treating a diseasestate involving a leczyme, by administering an effective amount of acarbohydrate ligand that binds to the leczyme. As used herein, the term“disease state” includes any diseases, whether genetic or acquired,provided a leczyme plays a role in the disease process. Such diseasestates include inflammation, transplantation rejection, and alsoincludes diseases having both a genetic and an environmental basis suchas iron storage diseases, autoimmunity or cancer. In addition, a diseasestate includes diseases resulting from an infectious agent such as avirus, bacteria, yeast or parasite. The ability of an infectious agentto enter and infect cells of the host can occur by binding to leczyme orcarbohydrate ligand expressed on the cells of the host. A peptidemimetope for a carbohydrate can also be used to treat a disease statethat involves a leczyme for which the mimetope can bind.

[0090] The present invention provides methods for treating a diseasestate involving a leczyme by administering a leczyme having a similarbinding specificity for a carbohydrate ligand as the leczyme involved inthe disease state. The disease states useful for treatment by a leczymeinclude those described above for treatment by a carbohydrate ligand.Thus, aberrant self-recognition, mediated by a leczyme in a diseasedindividual, can be treated by administration of a leczyme. Such aleczyme can bind to the natural carbohydrate ligand detected on a targetcell by the aberrant self-reactive leczyme-expressing cell, and,therefore, block the ability of the self-reactive leczyme-expressingcell to recognize and react aberrantly towards the target cell.

[0091] The present invention provides methods for treating an ironmetabolic disorder known as hemochromatosis. Defects in iron metabolismcan have a basis in leczyme function. In elevated concentrations, ironis a toxic inorganic molecule that has been implicated in thepathophysiology of a number of common diseases. These include but arenot limited to cancer (Stevens et al, N. Engl. J. Med., 319:1047 (1988);Stevens, et al., Med. Oncol. Tumor Pharmacother, 7:177-181 (1990)),heart disease (Kannel, et al, 1976; Sullivan, Lancet, 1:1293-1294(1981); Salonen, et al, Circulation, 36:803-811 (1992)), reperfusioninjury (Zweier, J. Biol. Chem., 263:1353-1357 (1988)) and rheumatoidarthritis (Blake et al., Arthritis Rheum., 27:495-501 (1984)). There isno argument that severe iron overload results in a constellation ofpathologies, collectively called hemochromatosis, the most commongenetic disease affecting man.

[0092] Hemochromatosis results from enhanced absorption of iron from theGI tract by active transport but the underlying metabolic defect iscurrently unknown. Identification of the genes responsible for theabsorption of iron, and developing an animal model in which ironoverload is due to active enhanced absorption of iron from the GI tract,would greatly facilitate understanding hemochromatosis and increaseknowledge about the general mechanisms of iron metabolism. The presentinvention provides the results from a new animal model and data fromhumans that indicate a role for an MHC-encoded leczyme in thepathogenesis of hemochromatosis.

[0093] Hemochromatosis is not usually brought to clinical attentionuntil symptoms develop, and several studies have indicated that removalof the iron after the development of tissue damage does not necessarilyimprove the organ function (Cundy, et al., Clin. Endocrinol., 38:617-620(1993); Westera et al., Am. J. Clin. Path., 99:39-44 (1993)).Hemochromatosis is an underdiagnosed and undertreated disease that wouldbenefit greatly from early diagnosis and an effective treatment (forreviews see Edwards et al., Hosp. Pract. Suppl., 3:30-36 (1991); Edwardsand Kushner, N. Engl. J. Med., 328:1616-1620 (1993)).

[0094] Untreated hemochromatosis is characterized by iron overload ofparenchymal cells, which is toxic and the probable cause of variouscomplications including hepatopathy (including cirrhosis, and livercancer), arthropathy, hypogonadotropic hypogonadism, marrow aplasia,skin disorders, diabetes mellitus, and cardiomyopathy (for review seeHalliday and Powell, Iron and Human Disease, Lauffer, R B, (ed). 131-160(1992)). There are reportedly 1.5 to 2 million active cases ofhemochromatosis within the U.S., with approximately 25% of latediagnosed or untreated patients developing hepatomas.

[0095] In untreated hemochromatosis, iron is universally deposited inthe hepatocytes of the liver, and elevated saturation of transferrinwith elevated serum ferritin levels combined with liver biopsy providesthe best diagnostic test currently available (Fairbanks, Hosp. Pract.,26:17-24 (1991)). The iron is found primarily in the cytoplasm ofhepatocytes, and by electron microscopy in lysosomal vacuoles, and inmore severe cases, iron is deposited in mitochondria (for review seeIancu, Ped. Pathol., 10:281-296 (1990)). Other liver toxins such asalcohol and hepatitis exacerbate the damage caused by the irondeposition (Piperno et al., J. Hepat., 16:364-368 (1992)). Patients withhemochromatosis are advised not to drink alcohol, because of increasedliver damage, or to smoke tobacco products, as iron deposition can alsooccur in the lungs.

[0096] Hemochromatosis is an autosomal recessive disease in which theresponsible gene(s) is ligand to the A locus of the human MHC (HLAcomplex), located on human chromosome 6 (Simon and Brissot, Hepatol.,6:116-124 (1988)). Linkage to human HLA-A3 has been documented inapproximately 73% of cases. However, other genetic loci also have beenimplicated, especially in African (Gorduke et al., N. Engl. J. Med.,326:95-100 (1992)) and African-American populations (Barton et al.,Blood, 35:95a (1993))

[0097] Hemochromatosis is the most common genetic malady in humans farexceeding cystic fibrosis, phenylketonuria and muscular dystrophycombined (Leggett et al., Clin. Chem., 36:1350-1355 (1990)). Oneexplanation for the high incidence of this genetic disease may be thatresults from different mutations in multiple linked genes that producesa similar phenotype. Hemochromatosis occurs most frequently inpopulations of European origin with a frequency in homozygotes andheterozygotes of approximately 0.3 and 13%, respectively.

[0098] Several markers, including the recently described D6S105, havebeen identified in the human MHC locus and have narrowed the genomiclocation of the hemochromatosis gene to within 1 centimorgan of the Alocus (Jazwinska et al., Am. J. Hum. Genet., 53:347-352 (1993)), andpossibly centromeric to HLA-F (Gasparini, et al., Hum. Mol. Genet.,5:571-576 (1993)). Others have reported candidate (HC) genes located20-200 kb telomeric to HLA-A (el Kahloun et al., Hum. Mol. Genet.,2:55-60 (1993)). While several of these candidate genes were thought tobe single copy, three of the genes, termed HCG II, IV and VII, werefound to be multicopy genes. Thus, despite the advances made indetermining the location of the HC gene, it has not yet been isolated.

[0099] Animal models for iron overload exist, however, these models arenot entirely suitable for the study of hemochromatosis since they do notreflect enhanced iron absorption from the gut by active transport. Micehomozygous for deletion of the gene encoding β₂M (β₂−/−mice (Koller etal., Science, 248:1227-1230 (1990); Zijlstra et al., Nature, 344:742-746(1990)) provide an excellent animal model for the study orhemochromatosis. These animals lack detectable class I proteins on thecell-surface, although biochemical labeling shows that class I geneproducts are being synthesized. Activated lymphocytes from β₂−/− animalscan be lysed by activated natural killer (NK) cells, again suggesting adeficiency in class I expression (Liao et al., Science, 253:199-202(1991)). These mice were originally developed to study the role of β₂Min development. While the mice developed and bred normally, they failedto generate significant numbers of CD8+ T cells. Consequently, thesemice have been intensely studied from an immunologic perspective.

[0100] β₂−/−mice combat viral infections relatively well, although thecourse of the infection is longer than in normal animals (Eichelbergeret al., J. Exp Med., 174:875-878 (1991); Muller et al., Nature,255:1576-1579 (1992)). They reject allografts (Zijlistra et al., J. Exp.Med., 175:885-889 (1992)) and show higher levels of Ig production andfaster class switching of antibody types than normal mice. Although CD8+T cells are low to undetectable at birth, studies have shown that theanimals can generate CD8+ T cells, and a cytotoxic CD8+ T cell responsecan be mounted under appropriate circumstances (Apasov and Stikovsky, J.Immunol., 152:2087-2097 (1994). Another significant abnormality reportedin these animals is that they develop hyperglycemia (glucose>300 mg/dl)in old age (greater than 2 years), It has been suggested that the onsetof diabetes in the β₂−/− mice is related to autoimmunity (Faustman etal., Science, 254:1756-1761 (1991)), however this explanation has beendisputed (Serreze et al., Diabetes, 43:505-509 (1994); Wicker et al.,Diabetes, 43:500-504 (1994)).

[0101] β₂−/−mice can develop iron overload that is similar to humanhemochromatosis. β₂−/−mice can spontaneously develop hepatomas. Thisobservation combined with the molecular biology data of the β-GAP genes(see Example I), suggested that the mice would develop iron overload.Histochemical examination of tissues from these mice, confirmed thishypothesis. Iron was found deposited in the liver of all animals, and inthe kidneys, spleen and lungs of some of the animals. In addition, 16%of the animals developed liver disease, having either hepatomas or livernecrosis. Thus, the clinical findings for the β₂−/−deficient mice aresufficiently similar to the pathology of hemochromatosis to make theβ₂−/−mouse an attractive model for the study of a mechanism underlyinghuman hemochromatosis. More importantly, the β₂−/−mice demonstrate thatβ₂M plays a role in this disease.

[0102] The ability of β-GAP promoters to co-regulate both the β-GAP geneand a nonclassical class I gene that encodes leczyme, both of which areexpressed in the intestine, supports a role for a class I leczyme inhemochromatosis. The nonclassical class I gene regulated by the β-GAPpromoter is a leczyme that can recognize and modify a carbohydratestructure associated with the β-GAP gene product, the latter of whichdirectly or indirectly binds iron (ie. β-GAP can be an iron carrier).Disruption of β₂M expression results in a loss of regulation of theleczyme function provided by the nonclassical class I molecule, leadingto iron overload and hemochromatosis.

[0103] A carbohydrate ligand or a leczyme of the present invention canbe used to prepare a medicament for the treatment of a disease statesuch as hemochromatosis, autoimmune disease, transplantation rejection,inflammation or infection. Autoimmune diseases that can be treated bythe present invention include systemic autoimmune diseases such asankylosing spondylitis, multiple sclerosis, rheumatoid arthritis,slceroderma, Sjögren's syndrome or systemic lupus erythematosus, andorgan-specific autoimmune diseases such as Addison's disease,Goodpasture's syndrome, Grave's disease, Hashimoto's thyroiditis,idiopathic thrombocytopenia purpura, myasthenia gravis or perniciousanemia. As hemochromatosis in humans is likely mediated by a β-GAPpromoter-driven leczyme, then treatment with a carbohydrate ligand,leczyme or competing molecule with the same or similar bindingspecificity as the leczyme involved in the disease can be used tomodulate the disease process. A carbohydrate ligand that binds thenonclassical class I leczyme involved in hemochromatosis can beadministered to inhibit binding to the β-GAP iron carrier.

[0104] A process to follow for using a carbohydrate ligand to treat adisease such as autoimmunity can first require identification of thelecyzme that is involved in the disease process. Subsequently, acandidate carbohydrate ligand that can bind to the leczyme is identifiedby methods disclosed herein. Thus, such candidate carbohydrate ligandscan then be tested in vitro to identify those efficient at blocking theautoimmune reaction exhibited when the leczyme on autoreactive immunecells from the diseased individual recognizes a carbohydrate moleculeexpressed on the cells of the individual that is the target of theautoreactive cell. The autoimmune reaction can be measured by anincrease in a cell function such as cell proliferation or release ofcytokines (see for example, Coligan et al. supra, 1992; Mishell andShiigi, supra, 1980). The best candidate carbohydrate ligands can thenbe used as a medicament to treat the disease.

[0105] The methods disclosed herein for the treatment of hemochromatosisare also suitable for the treatment of many other medical diseases orcomplication resulting from iron overload. Since multiple leczyme genesare involved in mediating control of iron metabolism, the type ofmutation, its location in the gene and the number and type of leczymegenes mutated in an individual are factors that can effect the extent ofiron overload in an individual. As the extent of iron overload exhibitedby an individual is dependent on the above factors, then the methodsdisclosed herein to treat hemochromatosis are also applicable fortreating other diseases resulting from iron overload. Such diseasesinclude, for example, hepatopathy (including cirrhosis, and livercancer), arthropathy, hypogonadotropic hypogonadism, marrow aplasia,skin disorders, diabetes mellitus, and cardiomyopathy (for review seeHalliday and Powell, Iron and Human Disease, Lauffer, R B, (ed). 131-160(1992)).

[0106] In order to modulate hemochromatosis or other iron storagedisease, the carbohydrate ligand or mimetope is administered in aneffective amount. The total effective amount can be administered to asubject as a single dose, either as a bolus or by infusion over arelatively short period of time, or can be administered using afractionated treatment protocol, in which the multiple doses areadministered over a more prolonged period of time. One skilled in theart would know that the concentration of carbohydrate ligand required toobtain an effective dose in a subject depends on many factors includingthe age and general health of the subject as well as the route ofadministration and the number of treatments to be administered and thechemical form of the carbohydrate ligand. In view of these factors, theskilled artisan would adjust the particular amount so as to obtain aneffective amount for the subject being treated.

[0107] A carbohydrate ligand or a leczyme also is useful in vivo for thetreatment of autoimmune diseases involving a leczyme. In autoimmunedisease, a leczyme expressed on a lymphoid cell can recognize aself-carbohydrate ligand as foreign carbohydrate ligand, resulting inimmune-directed destruction of cells expressing the self-carbohydrateligand. Thus, administration of a carbohydrate ligand, mimetope or othercompeting molecule that can bind to the leczyme involved in aberrantself-recognition can block lymphoid cell recognition or activationleading to a reduction in symptoms or cessation of autoimmune disease.Alternatively, administration of a leczyme that has the same or similarbinding specificity for the self-carbohydrate ligand recognized by aleczyme of the autoreactive lymphoid cell can also be used to treat theautoimmune disease.

[0108] A carbohydrate ligand or a leczyme can be used to treat a diseasestate resulting from an infectious agent such as a virus, bacterium,yeast or parasite. Infectious agents have evolved to express their ownexternal receptors that can recognize carbohydrate structures orleczymes on the cell-surface, enabling entry of the agent into the cellto be infected. Thus, administration of an appropriate carbohydrateligand or a leczyme to an individual exposed to an infectious agent canblock the binding of the agent to target cells, subsequently inhibitingthe extent of infection and thereby reducing the spread of the disease.

[0109] A carbohydrate ligand or a leczyme of the present invention canbe used to treat transplantation rejection. Since rejection is based onthe recognition of foreign molecules by lymphocytes of the transplantrecipient, then treatment with a carbohydrate ligand that can bind tothe leczyme of the transplant recipient's lymphocyte that is involved inforeign antigen recognition can inhibit recognition leading totransplantation rejection. Also, administration of a leczyme that hasthe same or similar binding site specificity as the leczyme of atransplant recipient's lymphocyte involved in foreign antigenrecognition can inhibit recognition leading to transplantationrejection.

[0110] A carbohydrate ligand or leczyme of the present invention isparticularly useful when administered as a pharmaceutical compositioncontaining a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers are well known in the art and include, for example,aqueous solutions such as a physiologically buffered saline or othersolvents or vehicles such as glycols, glycerol, oils such as olive oilor injectable organic esters.

[0111] A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize or to increasethe absorption of a carbohydrate ligand or leczyme. Such physiologicallyacceptable compounds include, for example, carbohydrates, such asglucose, sucrose, dextrans, antioxidants, such as ascorbic acid orglutathione, chelating agents, low molecular weight proteins or otherstabilizers or excipients. One skilled in the art would know that thechoice of a pharmaceutically acceptable carrier, including aphysiologically acceptable compound, depends, for example, on the routeof administration of the composition.

[0112] One skilled in the art would know that a pharmaceuticalcomposition containing a carbohydrate ligand or leczyme can beadministered to a subject by various routes including, for example, bydirect instillation, orally or parenterally, such as intravenously,intramuscularly, subcutaneously or intraperitoneally. The compositioncan be administered by injection or by intubation. The pharmaceuticalcomposition also can be incorporated, if desired, into liposomes ormicrospheres or can be microencapsulated in other polymer matrices(Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton, Fla.,1984), which is incorporated herein by reference). Liposomes, forexample, which consist of phospholipids or other lipids, are nontoxic,physiologically acceptable and metabolizable carriers that arerelatively easy to make and administer.

[0113] An expression vector encoding a leczyme can be administered invivo to treat a disease state resulting from a leczyme. For example, adisease state resulting from a mutated leczyme, such as anemia, can betreated by administering an expression vector encoding a functionalleczyme involved in iron transport and obtaining expression of thevector in cells of the digestive tract.

[0114] The level of expression of a particular leczyme in a cell canhave a impact on the nature of a carbohydrate ligand expressed by thecell. If expression of a particular carbohydrate ligand is involved in adisease process, the ligand can be eliminated from a cell by reducingthe expression of the leczyme responsible for producing the ligand.Thus, an expression vector can contain an exogenous nucleic acidmolecule encoding an antisense nucleotide sequence that is complementaryto a nucleotide sequence encoding a portion of a leczyme such that whenintroduced into a cell under the appropriate conditions, the expressionvector can produce an antisense nucleic acid molecule, which canselectively hybridize to the leczyme gene or message in a cell and,thereby, affect the expression of the leczyme in the cell. For example,the antisense nucleic acid molecule can hybridize to a leczyme gene inthe cell and can reduce or inhibit transcription of the leczyme gene.Also, the antisense molecule can hybridize to the message encoding theleczyme in the cell and can reduce or inhibit translation, processingand cell stability or half-life of the RNA.

[0115] Expression vectors also can be used to effect the expression of aleczyme or of a carbohydrate ligand involved in a disease state byintroducing into a cell an exogenous nucleic acid molecule encoding aribozyme that can specifically cleave RNA encoding the leczyme orpeptide backbone of a carbohydrate ligand. Thus, by introducing theribozyme into cells involved in a disease process, one can reduceexpression of the leczyme or carbohydrate ligand involved in the diseaseand therefore reduce or inhibit the disease process. An antisensenucleic acid molecule or a ribozyme can be chemically synthesized andincorporated into an expression vector using recombinant DNA techniques.The antisense nucleic acid molecule or ribozyme also can be addeddirectly to a cell without having been incorporated into an expressionvector.

[0116] Methods for introducing an expression vector into cell are wellknown in the art. Such methods are described in Sambrook et al, supra,1989; Kriegler M. Gene Transfer and Expression: A Laboratory Manual (W.H. Freeman and Co. New York, N.Y. (1990), both of which are incorporatedherein by reference) and, for example, include transfection methods suchas calcium phosphate, electroporation, lipofection, or viral infection.

[0117] Recombinant viral rectors are available for introducing anexogenous nucleic acid molecule into a mammalian cell and include, forexample, adenovirus, herpesvirus and retrovirus-derived vectors. Forexample, a viral vector encoding a leczyme can be packaged into a virusto enable delivery of the genetic information and expression of theseleczyme in gastrointestinal epithelial cells following infection by thevirus. Also, a recombinant virus which contains an antisense sequence ora ribozyme specific for a nucleotide sequence encoding a leczyme canintroduced into a cell in an individual to inhibit a disease statemediated by the leczyme or a leczyme with a similar carbohydrate bindingspecificity.

[0118] Recombinant viral infection can be more selective than direct DNAdelivery due to the natural ability of a virus to infect only certaintypes of cells. This natural ability for selective viral infection canbe exploited to limit infection to only certain cell types within amixed cell population. For example, adenoviruses can be used to restrictviral infection principally to cells of epithelial origin. In addition,a retrovirus can be modified by recombinant DNA techniques to enableexpression of a unique receptor or ligand that provides furtherspecificity to viral gene delivery. Retroviral delivery systems thatprovide high infection rates, stable genetic integration and high levelsof exogenous gene expression are well known in the art.

[0119] As described above, recombinant viral delivery systems exist thatprovide the means to deliver genetic information into a selected type ofcell. The choice of viral system will depend on the desired cell type tobe targeted, while the choice of vector will depend on the intendedapplication. Recombinant viral vectors are readily available to those inthe art and can be easily modified by one skilled in the art usingstandard recombinant DNA methods (see, for example, Krieger, GeneTransfer and Expression: A Laboratory Manual, (W. H. Freeman. andCompany, 1990); Goeddel, Methods in Enzymology, vol. 185, (AcademicPress, 1990); and Stoker, In Molec. Virol., A Practical Approach (eds.Davison and Elliott, IRL Press, 1993), all three of which areincorporated herein by reference).

[0120] The present invention provides methods for diagnosing a geneticpredisposition for hemochromatosis or other iron storage diseases basedon a leczyme by detecting a mutation in the heavy chain of a class I MHCmolecule encoded for by a gene in the MHC locus. These methods can beused to diagnose an individual having the symptoms of an iron storagedisease. A positive diagnosis of mutation in an individual's heavy chainis useful to verify the underlying cause of the disease and byidentifying the particular leczyme that is mutated. The identificationof the mutated leczyme can be used with the methods disclosed herein toidentify a carbohydrate suitable for treating the disease.

[0121] An individual who does not have an iron storage disease, but issuspected of inheriting a mutation that can predispose the individual todevelop an iron storage disease later in life can also benefit fromhaving their class I molecules tested for mutation by the methodsdisclosed herein.

[0122] A mutation that is diagnostic for the disease is one that resultsin a significantly reduced affinity of the heavy chain for human β₂M.For example, a mutation in a nonclassical class I heavy chain thatresults in deletion of a signal for phosphorylation is a mutation thatis diagnostic for hemochromatosis since a properly phosphorylated heavychain is necessary for the chain to interact with β₂M. Consensus aminoacid sequences that signal a cell to phosphorylate a serine or athreonine residue in a polypeptide are well known in the art. A mutationthat is diagnostic for hemochromatosis also can occur in a region of theheavy chain that is near to a phosphorylation site. Such a mutation canreduce the ability of the heavy chain to associate with β₂M if thephosphate group added to this site cannot be removed in a cell.

[0123] Methods to detect a phosphorylation site mutation in anonclassical class I heavy chain can be based either on analysis of theprotein or the nucleic acid encoding the protein. For proteindetermination, the nonclassical class I molecule can be purified from asource of cells or body fluids of an individual and the heavy chain canbe isolated from β₂M. Methods to purify a class I MHC molecule andisolate the heavy chain from β₂M are well known in the art. The isolatedheavy chain can then be subjected to amino acid sequencing, peptidemapping or other such protein analyses to determine if the sequence aphosphorylation site has been mutated. Such methods for proteindetermination are well known to those in the art.

[0124] A mutation in a nucleic acid sequence can be detected by variousmethods to analyze nucleic acids such as by nucleic acid sequencing,polymerase chain reaction or hybridization. Such methods are well knownto those in the art (see, for example, Sambrook et al, supra, 1989;Hames and Higgins Nucleic Acid Hybridisation: a practical approach (IRLPress, New York, 1985), both of which are incorporated herein byreference).

[0125] Methods to detect decreased binding of a mutated heavy chain withβ₂M can be used for diagnosing an iron storage disease such ashemochromatosis. In these methods, the heavy chain of an class I MHCmolecule is isolated from an individual and contacted with β₂M underconditions suitable for a non-mutated such heavy chain to associate withβ₂M. A control reaction, which contains a non-mutant form of the same orsimilar class I heavy chain to the one being tested for a mutation isperformed in parallel. After contacting the heavy chain with β₂M, thereaction is incubated under suitable conditions, including, for example,an appropriate buffer concentration and pH and time and temperature,which is sufficient for the control heavy chain to associate with β₂M.The heavy chain being tested from the individual is considered to have amutation diagnostic for an iron storage disease when the fraction ofthis heavy chain that associates with β₂M is significantly less than thefraction of control heavy chain that associates with β₂M.

[0126] The association of a class I heavy chain with β₂M can bedetected, for example, by attaching one of the molecules to a solidsupport and attaching a detectable label such as a radionuclide or afluorescent label to the other molecule and measuring the amount ofdetectable label that is associated with the solid support, wherein theamount of label detected indicates the amount of association of theheavy chain with β₂M.

[0127] The following examples are intended to illustrate but not limitthe present invention.

EXAMPLE I Cloning and Expression of the β-GAP Genes

[0128] This example provides an approach to identify and clone leczymegenes from various species of animal to elucidate their role in ironmetabolic diseases.

[0129] Cloning of the Mouse β-Gap Genes

[0130] Genomic λ libraries were constructed by partial Hae III digestionof DNA from A/J and Balb/c mouse liver and cloning the fragments intothe vector Charon 4A. The libraries were screened with the S15 probe,isolated from the H-2L^(d) gene (Margulies et al., Nature, 295:168-170(1982), which is incorporated herein by reference)). S15 is a 3′ class IMHC mouse probe and consists of 522 base pairs including 36 base pairsof exon 4 encoding the alpha-3 domain and 486 base pairs of intron(Evans et al., Proc. Natl. Acad. Sci. (USA), 79:1994-1998 (1982)).Probes were prepared by excising the insert from M13 RF or pUC18,purifying the fragment from disulfide cross-linked acrylamide gels(Hansen, Anal. Biochem. 116:146-151 (1981)), and labeling with ³²P to aspecific activity of >10⁸ cpm/μg by nick translation (Rigby et al., J.Mol. Biol. 113:237-251 (1977)). Libraries were screened using standardcolony hybridization techniques (for details see Sambrook et al., supra,1988).

[0131] Seventeen unique λ clones were isolated from the libraries andwere subjected to restriction enzyme digestion mapping. BamHI digestionand gel electrophoresis of these clones revealed five from the A/Jstrain and one from the Balb/c stain that contained a unique 500 basepair (bp) BamHI restriction fragment (BB500). The six clones containingthe unique fragment were subjected to BAMHl digestion, the BB500fragment was gel purified and subcloned into M13 vector Mp18 and mp19(Yanisch-Perron et al., Gene 33:103-119 (1985)). DNA sequences weredetermined by the chain termination method (Sanger, et al., Proc. Natl.Acad. Sci. (USA) 74:5463-5467 (1977)) using ³⁵S-ATP. Reactions wereanalyzed on 6% urea-polyacrylamide gradient gels (Biggins et al., Proc.Natl. Acad. Sci. (USA) 80:3963-3965 (1983)). DNA sequences wereassembled and analyzed using the University of Wisconsin Computer GroupPrograms (Devereux, et al., Nucleic Acids Res. 12:387-395 (1984)) run ona VAX-11/785 computer.

[0132] DNA sequence comparisons demonstrated that the BB500 fragmentsshare greater than 93% sequence homology. A region within the BB500fragment shows 100% sequence homology between the λ clones and has beentermed β-GAP (globin analogous promoter) since it is a regulatory motifthat shares sequence homology with mouse, rabbit or human β-globinpromoters (for a detailed comparison see ahead). There is closesimilarity between all six fragments (called β-GAP1-6) with the minorexception of β-GAP4 where an 8 base pair sequence AAGAGGAG, immediatelydownstream of a CCAAT element, has been deleted. There are other minordifferences between these sequences, and the λ clones they have beenisolated from demonstrate different restriction patterns confirming thatthe various λ clones contain unique sequences and are not a cloningartifact. Thus, the A/J strain mouse contains at least five highlyhomologous β-GAP sequences within its genome.

[0133] Mapping the β-GAP Sequences Map to Chromosome 17 in the Mouse

[0134] Southern blotting was used to determine if the BB500 sequencecould identify genes located on chromosome 17 of the mouse. DNA fromseveral Chinese hamster ovary (CHO) mouse somatic hybrid cell lines wereevaluated by Southern blotting using the β-GAP6 BB500 probe. Genomic DNAwas isolated from cultured cells, digested with EcoR1, electrophoresedon 0.8% agarose gels and transferred to a nitrocellulose membrane.Hybridization with the BB500 probe was carried out in the presence ofdextran sulfate under the conditions described by Meinkof and Wahl(Anal. Biochem. 138:267-284 (1985)) with a final wash in 0.2× sodiumchloride sodium citrate buffer, pH 7.0 (SSC) at 60° C.

[0135] The BB500 probe hybridized with the HM27 cell line containing theDNA from mouse chromosomes 15 and 17 and revealed the same bandingpattern as with total genomic BALB/c DNA. The cell line HM65 that lacksBALB/c chromosome 17 was devoid of hybridizable bands, indicating thatthe probe did not bind nonspecifically to CHO DNA. DNA from other CHOcell lines containing mouse chromosomes other than chromosome 17 wereexamined by Southern blotting with the BB500 probe and were found to benegative (not shown). These results indicate that the β-GAP sequencesall map to chromosome 17 in the mouse.

[0136] Mapping the β-GAP Sequences to the Murine Q/TL Complex

[0137] The fact that the β-GAP sequences were isolated from the mousegenome provided several powerful tools to precisely map the location ofthe sequences. First, the murine MHC is highly characterized,particularly with respect to the nonclassical class I region and,secondly, congenic strains of mice exist where the position of genes inthe MHC can be pinpointed. Congenic strains were originally developed bybreeding strains of inbred mice together. Subsequent generations ofchromosomal crossing over has produced a number of strains which containa portion of the MHC from one strain and the remainder of the MHC fromanother strain. Consequently, it is possible to compare restrictionfragment length polymorphism (RFLP) between the strains, and determineif the banding patterns are linked to a given MHC locus (for review seeKlein, Natural History of the Major Histocompatibility Complex, 50-73(1986)). RFLP analysis was performed by obtaining purified genomic DNAfrom the various mouse strains, digesting the DNA with EcoRI andperforming Southern hybridization with the β-GAP6 BB500 probe asdescribed above. The Southern blot showed that the probe identified upto ten different bands from the DNA of the mouse strains tested (Table1). Four of these bands, 30 kb, 20 kb, 16 kb and 10.5 kb, were mappedwithin the MHC locus. The RFLP analysis indicated that there were atleast four to six copies of the β-GAP sequences/genome depending on thestrain of mouse tested. In addition genetic analysis of the RFLPpatterns indicated that the 30 kb and 10.5 kb β-GAP bands mapped to Qregion between Q1 and Q4 while the 20 kb and 16 kb β-GAP bands mapped tothe T region. In addition, two of the β-GAP sequences that did notdemonstrate RFLP polymorphism were mapped telomeric to the classicalclass I genes.

[0138] Locating the β-GAP Sequences Directly Adjacent to NonclassicalClass I Genes

[0139] The two of the β-GAP gene sequences that were mapped to the Qregion between Q1 and Q4, were directly linked to Q1 and Q2 by DNAsequence analysis of Q1 and Q2 genes isolated from a C57BL/6 (H-2°) λlibrary. Sequencing showed that both the Q1 and Q2 genes are associatedin a head to head configuration with an unknown gene (currently definedas the β-GAP gene) with both genes transcriptionally regulated by asingle promoter/enhancer region having two promoters defined by a pairof CAAT and TATA boxes located about 25 bp apart on opposite strands ofthe DNA. Thus, having intact promoters and a common regulatory region,the class I and β-GAP genes would be transcribed from opposite strands,with the class I genes Q1 or Q2 transcribed from 5′ to 3′ on the topstrand and the β-GAP gene transcribed from 5′ to 3′ on the bottomstrand.

[0140] The sequence analysis of Q1 and Q2 genes from C57BL/6, as well asa TL gene from A/J (H-2^(a), Watts et al. EMBO J. 8:1749-1759 (1980))indicated that β-GAP promoter and regulatory regions had replaced thetypical classical class I-type 5′ regulatory sequences known to beinvolved in the regulation of classical class I genes. The β-GAPpromoter is an active promoter since it is known that the Q2 geneexpresses a gene product that can be detected in the intestine (Wang etal., Immunogenet., 38:370-372 (1993)). These results indicate that theβ-GAP promoter regulates the expression of some nonclassical class Igenes. TABLE 1 COMPARISON OF SOUTHERN BLOT ANALYSIS OF EcoRI DIGESTS OFMURINE DNA USING THE BB500 LOW COPY NUMBER PROBE WITH GENETIC MAPS OFVARIOUS ALLOGENEIC AND CONGENIC STRAINS. MHC REGION EcoRI BAND SIZE (kb)STRAIN K D Q T 30 20 16 14.5 13 10.5 9.2 8.0 7.8 7.5 B6, B10 b b bb + + + + + B6, K1 b b k k + + + + + B6, K2 b b bk k + + + + + AKR k k kk + + + + + B6, K3 k k b a + + + + B6, K4 k k k a + + + + + B6-H-2^(k) kk k k + + + + + B6-T1a^(a) b b ba a + + + + + A/J k d a a + + + + + +Balb/cJ d d d d + + + + + + B10, A k d a a + + + + + + A-T1a^(b) k d ab + + + + +

[0141] Head to head gene structure with co-regulation of the genes hasbeen previously described in organisms ranging from bacteria to humans,indicating that co-regulation is a widely adopted strategy. (Brickman etal., J. Molec. Biol., 212:669-682 (1990); Xu and Doolittle, Proc. Natl.Acad. Sci. (USA), 87: 2097-2101 (1990); Lennard and Fried, Molec. Cell.Biol., 11:1281-1294 (1991); Heikkila et al., J. Biol. Chem.,268:24677-24682 (1993); Fererjian and Kafatos, Dev. Biol., 161:37-47(1994); Sun and Kitchingman, Nucleic Acids Res., 22:861-868 (1994)). Inboth prokaryotic and eukaryotic systems, interaction between, or linkagein a metabolic pathway of two gene products has been suggested(Galvalas, et al., Mol. Cell. Biol., 13:4784-4792 (1993); Lightfoot etal., Br. J Cancer, 69:264-2673 (1994)). It should be noted that in theβ-GAP clones so far studied, the Q1 and Q2 genes still possesses theirown CAAT and TATA elements, and it is only the typical classical class Iregulatory enhancer regions which are absent.

[0142] Conservation of the β-GAP Sequences Across Species

[0143] To demonstrate that the β-GAP sequences are conserved, and thatvarious species, including human, contain multiple copies of these genesa “Zoo blot” of various species of genomic DNAs was digested with EcoRIand analyzed by Southern blotting using the murine β-GAP6 BB500 probe.Under low stringency the blot showed detection of a multiplicity ofbands in DNA from human, rat, mouse, dog, rabbit and monkey. Thisindicates that multiple copies of the β-GAP sequences were found in manyspecies including human. In addition, the conservation of the β-GAPmultigene family predates speciation of murine and human and thereforeis not the product of a recent gene duplication or rearrangement. Thedemonstration of interspecies sequence homology is significant because,in general, exons and regulatory regions tend to be conserved. Thus, thepattern of specific regions of retained homology suggests that the β-GAPsequences are retained by selective pressure.

[0144] Homology Between the β-GAP Sequences and the Promoters forβ-Globin

[0145] Sequences within all six of the 500 bp β-GAP clones show strikingsequence and positional homology to mouse, rabbit and human β-globinpromoter regulatory elements. Important regulatory elements within a 106bp region of the β-globin promoter have been characterized (Myers etal., Science 232:613-618 (1986); Stuve and Myers, Mol. Cell Biol.6:3350-3358 (1990)). Using saturation mutagenesis and 5′ deletionpromoters, Myers and his colleagues constructed a series of mutants thatwere used to identify four regulatory sequences. The four regulatorymotifs were located between positions −95 and −26 which contain a CACCCelement (positions −95 to −87), CCAAT and TATA box motifs at positions−79 to −72 and −30 to −26, respectively, and a 11 bp repeat elementlocated between the CCAAT and TATA boxes (positions −53 to −32) thatcontains 2 imperfect duplicated repetitive elements (βDRE). The factthat these βDRE are essential for the expression of globin genes hasbeen shown by deletional studies.

[0146] Comparison of the six 500 bp β-GAP sequences with the β-globinpromoter sequences from various species showed several striking sequencehomologies to β-globin regulatory elements. Analysis of the β-GAPsequence in this region revealed 5 regulatory motifs found in theβ-globin promoters. These include the 5′ CACCC erythroid element betweenpositions −127 to −123, CCAAT and TATA box motifs between positions −109to −105 and −30 to −26, respectively, the cap consensus sequencepositions −13 to −10, and a fifth and more complex regulatory elementinvolved a β-globin βDRE of a 10 and 11 bp sequence (base pair numberingwas determined from sequence alignments with gaps inserted and does notreflect the true base pair position from the transcriptional startsite).

[0147] In all the β-GAP clones, two of the four βDRE regulatory motifswere flanked by the CCAAT and TATA elements between positions −54 and−32, while two other βDREs were found immediately upstream of the TATAbox (positions −11 to +1 and +3 to +12). All of these βDRE wereconserved in sequence, and moreover, two of them were conserved inposition (−54 and −32). It is significant that the βDREs conserved inβ-GAP were conserved in globins from multiple species (mouse, rabbit,chicken and human) covering more than 100 million years of evolution.This observation of evolutionary conservation indicates the β-GAP genesare old genes.

[0148] A final putative regulatory motif from the β-GAP clones wasAGATAA (nucleotides −82 to −77), which is identical to the DNA consensussequence for the transcriptional binding factors NF-E1. This family ofDNA binding proteins (NF-E1a, b, and c) are involved in the erythroidand/or T-cell specific expression of many genes, including mouse andchicken adult β-globin, the heme pathway enzyme porphobilinogen (PGB)deaminase, the T-cell receptor and the leukemia virus HTLV III.

[0149] A closer inspection of the regions of homology between the β-GAPand mouse β-globin promoters reveals several features: 1) 18 of 26 basepairs match at positions −35 to −10 encompassing the consensus TATAmotif (Bucher, J. Mol. Biol. 212:563-578 (1990)); 2) a regionencompassing the β-GAP CCAAT box, positions −113 to −109 contains theβ-globin regulatory element CACCC which has been shown to be essentialfor the appropriate expression of β-globin in erythroid cells; 3) aperfect match of the CCAAT element exists at positions −109 to −105; 4)the fourth matching region encompasses a βDRE element, located betweenthe CCAAT and TATA boxes at positions −64 to −45 (this region contains16 of 19 bp matches with no gaps); and 5) a consensus cap site sequenceas defined by Bucher (Bucher, supra, 1990) and a putativetranscriptional start site is identified at nucleotides −13 to +1.

[0150] Several other putative regulatory sequences are apparent in theβ-GAP promoter. Between positions −68 and −37 and beginning 5nucleotides distal of the TATA element are 4 palindromes. The 5 basepair repeat TCAGA appears twice within 24 base pairs. These repeatsflank and are found within a globin-like imperfect direct repeat element(positions −57 to −47). Two longer palindromes with imperfect dyadsymmetry of 12 bp, and 15 bp, positions −67 to −56 and −51 to −37,respectively, contain smaller internal palindromes of 7 bp, CCTCAGG (−66to −60) and 5 bp repeat, TCAGA (−46 to −42), respectively. This β-GAP 33bp βDRE-like region combining the two large 12 and 15 bp imperfectpalindromes, the β-globin imperfect direct repeat element and the twoTCAGA palindromic repeats shows about 50% (16/33) nucleotide sequencehomology to the mouse β-globin promoter.

[0151] Expression of Genes Immediately Downstream from the β-GAPSequences in the Gastrointestinal Tract

[0152] The pattern of specific regions of retained homology between theβ-globin regulatory motifs and β-GAP promoters suggests: 1) thesequences have diverged from a common ancestral gene; and 2) thepreserved regions in the β-GAP sequences play a critical role in theregulation of expression of their respective genes. Furthermore, thehomology to promoters for genes intimately involved in iron metabolism,the occurrence of erythroid specific regulatory sequences, and the closeproximity of these genes to the human locus responsible forhemochromatosis, indicates a role for the β-GAP genes in ironmetabolism.

[0153] To demonstrate that the β-GAP promoters regulate downstreammessages, it is imperative to show that the associated genes encodetranscribable messages. Moreover, such messages should be expressed intissues involved in iron absorption, i.e. the gastrointestinal tract, ifthey are to be involved in the pathogenesis of hemochromatosis.

[0154] Northern blotting was performed with poly A+ RNA from variousorgans including the gastrointestinal tract. The blot was developedusing two probes derived from a β-GAP (Q2^(b)) cosmid clone. Totalcellular RNA was prepared by the TRIzol™ Reagent method according to themanufacturer's instruction (Gibco/BRL, Gaitherburg, Md.). poly A+ ormRNA was purified by oligo dT cellulose chromatography (Strategene, SanDiego, Calif.). RNA was analyzed on formaldehyde-agarose gels andtransferred to Zeta Bind membranes as previously described (Evans, etal., Proc. Natl. Acad. Sci. (USA) 81:5532-5536 (1984)). The cosmid clonecontaining a β-GAP sequence that was used or the probe was obtained froma λ library. The clone was digested with ApaLI and KpnI to yield a 10 kbfragment. The fragment was partially digested with BamHI to yield a 2 kbprobe encompassing the β-GAP sequences and a 8 kb probe piece furtherdownstream containing the coding sequences for a β-GAP gene.

[0155] Northern blotting with the 2 kb probe showed the presence ofpolydisperse messages produced in tissues from stomach, duodenum,jejunum, spleen and liver, principally of 5 kb and 8 kb in size. Thekidney showed less polydispersity with only the 8 kb band predominating.These results indicate that β-GAP promoter and upstream β-GAP codingsequences are expressed in the gastrointestinal tract and are associatedwith members of a multigene family of which the 5 Kb message of thejejunum is most prominent. The fact that this probe also recognized aband in the liver, spleen, kidney and stomach, suggested that relatedmembers of a β-GAP family can be functioning in other tissues. Thedownstream 8 Kb probe identified a band about 5 kb in jejunum which wasabsent in from the kidney polyA+ RNA. This result indicates thatdownstream β-GAP coding sequences are less conserved and can berestricted in expression.

[0156] The size and complexity of the β-GAP mRNA products detected bynorthern blotting is consistent with β-GAP genes coding for a family oflarge proteins. These characteristics are more like those of a mucinprotein family rather than an ion transport family of molecules. Thehomology to β-globin promoters, the occurrence of erythroid specificregulatory sequences and close proximity of nonclassical class I andβ-GAP genes to the locus responsible for hemochromatosis in humans, aninheritable disease of iron metabolism indicates a role for the β-GAPgenes and the nonclassical class I genes in iron metabolism. With thisinformation in hand and the facts disclosed herein that β₂M-knockoutmice have an unusually high incidence of hepatomas led to theunderstanding that these mice have a metabolic and pathologicalcondition similar to hemochromatosis.

[0157] Isolation of Murine β-GAP cDNAs From a Mouse Jejunal Library.

[0158] The 2 kb β-GAP cosmid probe was used to screen a mouse jejunalcDNA library (Strategene λ ZAP Express kit). Northern blots suggestedthat the messages recognized by the β-GAP probe were abundant (bandswere visible after only three hours of exposure) and this observationwas confirmed upon screening the library. Approximately 0.5% of theclones gave a positive signal on the initial screening. 30 positiveclones were picked and rescreened, and 26 positive clones were pickedfrom the secondary screen. The murine clones ranged in size fromapproximately 2 kb to >8 kb, and the size of the inserts corresponded tothe bands seen by northern blotting with the 2 kb probe.

[0159] DNA purified from the selected murine cDNA clones were digestedwith EcoRI and subjected to southern blot analysis. The blot was probedwith the 2 kb β-GAP cosmid probe, and 5 were found to be positiveindicating they contained β-GAP genes. These β-GAP clones are nearlyfull length cDNA since they were quite large and since they wereisolated with a 5′ β-GAP probe.

[0160] Cloning of the Human β-Gap Genes

[0161] A human genomic DNA library produced in sCOS cosmid vector wasprepared as described previously for producing a mouse genomic libraryin sCOS (Strategene, San Diego Calif.). The isolation of the human β-GAPgenes from the human sCOS cosmid library was performed by screeningclones with a class I MHC probe. The probe was generated from exons 4and 5 of the HLA-A2 gene, which encodes the highly conserved β₂M bindingdomain and the transmembrane region. Twenty five putative clonescontaining class I sequences were detected, and the Cosmids from theseclones were purified, cut with the restriction enzyme EcoRI, run on a0.7% agarose gel and blotted onto a charged nylon membrane. The blot washybridized with the class I probe, striped and rehybridized with the 2Kb β-GAP probe. Three unique clones were found that reacted with boththe murine β-GAP probe and the human class I probe. This resultindicates that the human β-GAP genes can be isolated and have a genomicstructure with a closely linked class I gene as was observed in mice andrats.

EXAMPLE II β₂M Knockout Mice Develop Iron Overload Similar toHemochromatosis

[0162] This example provides a method to analyze iron deficiency in ananimal model where an MHC-encoded leczyme function has been geneticallydeleted. In addition, these mice are useful for evaluating the in vivoutility of carbohydrate ligands on the treatment of hemochromatosis andvarious iron related diseases such as atherosclerosis, arthritis orcancer.

[0163] The data concerning iron overload in the β₂M knockout mice iscontained in Rothenberg and Voland, 1994. Histologic examination oftissues from 12-18 month old knockout mice, contained on a standarddiet, revealed evidence of hepatic necrosis. Iron stains of the tissuesrevealed iron deposition in the liver of all animals, and in the kidney,or the lung of approximately 10% of the animals. The standard dietcontains 350 mg/kg Ferric carbonate. When animals were placed on a“breeder diet”, which contains in addition to ferric carbonate, 10 mg/kgferrous sulfate, iron stores rose dramatically. Iron deposition in theanimals was also age related with the highest levels of iron seen in theoldest animals. Together these data indicate that the β₂M-knockout micedevelop iron overload that is diet and age related. In addition we haveshown that the animals develop hepatomas and others have reported thatolder animals develop diabetes (Faustman et al., Science 254:1756-1761(1991)). This constellation of pathologies mirrors humanhemochromatosis.

[0164] Although the invention has been described with reference to theexamples provided above, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the claims.

I claim:
 1. A composition, comprising a substantially purifiedcarbohydrate ligand that specifically binds to a leczyme.
 2. Thecomposition of claim 1, further comprising a polypeptide or lipid moietybonded to said carbohydrate ligand.
 3. A method of identifying acarbohydrate ligand that binds to a leczyme, comprising the steps of: a.contacting a sample containing a carbohydrate ligand with a leczymesuspected to bind such a ligand; and b. detecting whether binding of theligand and the leczyme has occurred.
 4. The method of claim 3, whereinsaid leczyme is an MHC-derived gene product.
 5. The method of claim 4,wherein said MHC-derived gene product is a class I molecule.
 6. Themethod of claim 4, wherein said MHC-derived gene product is a class IImolecule.
 7. The method of claim 4, wherein said MHC-derived geneproduct is an nonclassical class I molecule.
 8. The method of claim 3,wherein said leczyme is a non-MHC derived gene product.
 9. The method ofclaim 3, wherein said carbohydrate ligand is bonded to a polypeptide orlipid moiety.
 10. The method of claim 3, wherein said carbohydrateligand or said leczyme contains a detectable label.
 11. The method ofclaim 3, wherein said carbohydrate ligand or said leczyme is associatedwith a cell.
 12. A method of identifying a leczyme that binds to acarbohydrate ligand, comprising the steps of: a. contacting a samplecontaining a leczyme with a carbohydrate ligand suspected to bind such aleczyme; and b. detecting whether binding of the leczyme and the ligandhas occurred.
 13. The method of claim 12, wherein said leczyme is anMHC-derived gene product.
 14. The method of claim 12, wherein saidleczyme is a non-MHC derived gene product.
 15. The method of claim 12,wherein said carbohydrate ligand is bonded to a polypeptide or lipidmoiety.
 16. The method of claim 12, wherein said carbohydrate ligand orsaid leczyme contains a detectable label.
 17. The method of claim 12,wherein said carbohydrate ligand or said leczyme is associated with acell.
 18. A method of purifying a carbohydrate ligand that specificallybinds to a reagent, comprising the steps of: a. contacting a samplecontaining a carbohydrate ligand with a reagent capable of binding sucha ligand to form a ligand-reagent complex; b. separating the complexfrom the rest of the sample; and c. dissociating the ligand from thecomplex to obtain the purified ligand.
 19. The method of claim 18,wherein said reagent is a leczyme.
 20. The method of claim 18, whereinsaid reagent is an antibody.
 21. The method of claim 18, wherein saidleczyme is an MHC-derived gene product.
 22. The method of claim 18,wherein said leczyme is a non-MHC derived gene product.
 23. The methodof claim 18, wherein said carbohydrate ligand is bonded to a polypeptideor lipid moiety.
 24. A method of purifying a leczyme that specificallybinds to a carbohydrate ligand, comprising the steps of: a. contacting asample containing a leczyme with a carbohydrate ligand capable ofbinding such a leczyme to form a ligand-leczyme complex; b. separatingthe complex from the rest of the sample; and c. dissociating the leczymefrom the complex to obtain the purified leczyme.
 25. The method of claim24, wherein said leczyme is an MHC-derived gene product.
 26. The methodof claim 24, wherein said leczyme is a non-MHC derived gene product. 27.The method of claim 24, wherein said carbohydrate ligand is bonded to apolypeptide or lipid moiety.
 28. A method of identifying a carbohydrateligand that modifies the function of a leczyme-expressing cell,comprising the steps of: a. contacting a sample containing such acarbohydrate ligand with a leczyme-expressing cell; and b. subsequentlyassaying the cell to determine it's function.
 29. The method of claim28, wherein said leczyme is an MHC-derived gene product.
 30. The methodof claim 28, wherein said leczyme is a non-MHC derived gene product. 31.The method of claim 28, wherein said carbohydrate ligand is bonded to apolypeptide or lipid moiety.
 32. A method of identifying a leczyme thatmodifies the function of a carbohydrate ligand-expressing cell,comprising the steps of: a. contacting a sample containing such aleczyme with a carbohydrate ligand-expressing cell; and b. subsequentlyassaying the cell to determine it's function.
 33. The method of claim32, wherein said leczyme is an MHC-derived gene product.
 34. The methodof claim 32, wherein said leczyme is a non-MHC derived gene product. 35.A method of modifying the function of a leczyme-expressing cell,comprising contacting the cell with a carbohydrate ligand that binds theleczyme.
 36. The method of claim 35, wherein said leczyme is anMHC-derived gene product.
 37. The method of claim 35, wherein saidleczyme is a non-MHC derived gene product.
 38. The method of claim 35,wherein said carbohydrate ligand is bonded to a polypeptide or lipidmoiety.
 39. A method of modifying the function of a carbohydrateligand-expressing cell, comprising contacting the cell with a leczymethat binds the ligand.
 40. The method of claim 39, wherein said leczymeis an MHC-derived gene product.
 41. The method of claim 39, wherein saidleczyme is a non-MHC derived gene product.
 42. A method of identifying apeptide that binds to a carbohydrate ligand binding site of a leczyme,comprising the steps of: a. contacting a leczyme and a carbohydrateligand known to bind the leczyme with a test sample containing a peptideto be identified; b. determining the amount of carbohydrate ligand boundto the leczyme after said reacting; and c. comparing the amount ofcarbohydrate ligand bound in the test sample with the amount ofcarbohydrate ligand bound in a control sample, wherein a decreasedamount of carbohydrate ligand bound in the test sample relative to theamount of carbohydrate ligand bound in the control sample indicatesbinding of the peptide to the leczyme.
 43. The method of claim 42,wherein said peptide and said leczyme are contacted prior to contactingsaid carbohydrate ligand.
 44. The method of claim 42, wherein saidcarbohydrate ligand or said leczyme contains a detectable label.
 45. Themethod of claim 42, wherein said leczyme or said carbohydrate ligand isassociated with a cell.
 46. The method of claim 42, wherein said testsample contains a peptide library.
 47. A method of modifying a cell toproduce a carbohydrate ligand, comprising introducing an expressionvector encoding a leczyme into the cell to obtain expression of theleczyme, wherein said expression results in production of thecarbohydrate ligand by the cell.
 48. The method of claim 47, whereinsaid leczyme is an MHC-derived gene product.
 49. The method of claim 47,wherein said leczyme is a non-MHC derived gene product.
 50. A method formodulating an immune response in an individual to an antigen, comprisingadministering the carbohydrate ligand of claim 1 and the antigen. 51.The method of claim 50, wherein said administering results in anincrease in the immune response to the antigen.
 52. The method of claim50, wherein said administering results in a decrease in the immuneresponse to the antigen.
 53. The Method of claim 50, wherein saidadministering further comprises an immune suppressing agent.
 54. Themethod of claim 50, wherein said antigen and said carbohydrate ligandare covalently bonded.
 55. The method of claim 50, wherein an adjuvantis administered along with said antigen and said carbohydrate ligand.56. A method for treating a disease state involving a leczyme,comprising administering an effective amount of the carbohydrate ligandof claim
 1. 57. The method of claim 56, wherein said disease state is anMHC-linked disease.
 58. The method of claim 56, wherein said MHC-linkeddisease is an autoimmune disease.
 59. The method of claim 56, whereinsaid MHC-linked disease is hemochromatosis.
 60. The method of claim 56,wherein said disease state is an infection.
 61. The method of claim 56,wherein said disease state is transplantation rejection.
 62. A methodfor treating a disease state involving a leczyme, comprisingadministering an effective amount of a leczyme having a similar bindingspecificity for a carbohydrate ligand as the leczyme involved in thedisease state.
 63. The method of claim 62, wherein said leczyme is anMHC-derived gene product.
 64. The method of claim 62, wherein saidleczyme is a non-MHC derived gene product.
 65. The method of claim 62,wherein said disease. state is an MHC-linked disease.
 66. The method ofclaim 65, wherein said MHC-linked disease is an autoimmune disease. 67.The method of claim 65, wherein said MHC-linked disease ishemochromatosis.
 68. The method of claim 62, wherein said disease stateis an infection.
 69. The method of claim 62, wherein said disease stateis transplantation rejection.
 70. A method for detecting a geneticpredisposition for hemochromatosis, comprising detecting a mutation inthe heavy chain of a class I MHC molecule that reduces the ability ofsaid heavy chain to associate with β₂ microglobulin.
 71. The method ofclaim 70, wherein said mutation eliminates a signal for the addition ofa phosphate group.
 72. The method of claim 70, wherein said mutationeliminates the ability of a phosphate group in said heavy chain to bede-phosphorylated in a cell.
 73. A method for diagnosing hemochromatosisresulting from a reduction in the ability of a heavy chain of an MHCclass I molecule to bind β₂ microglobulin, comprising the steps of: a.isolating a class I MHC heavy chain from an individual to be tested; b.contacting the heavy chain with β₂ microglobulin under conditionssuitable for associating a class I MHC heavy chain with β₂microglobulin; and c. detecting the association of said heavy chain withsaid β₂ microglobulin, wherein a reduced association of said heavy chainwith β₂ microglobulin compared to the association of a control heavychain with β₂ microglobulin is diagnostic for hemochromatosis.